Health Articles

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Preliminary program

47TH SANDBJERG MEETING
ON MEMBRANE TRANSPORT
Monday May 18th - Wednesday May 20th 2015
Sandbjerg Estate
Sønderborg
Scientific program
MONDAY MAY 18TH

13.00-13.40
Arrival and coffee

13.40-13.45
Welcome and introductory remarks

SESSION 1

NA+/K+ ATPASE ISOFORMS IN HEALTH AND DISEASE
Coordinator: Karin Lykke-Hartmann and Bente Vilsen, Biomedicine, Aarhus, Denmark
13.45-14.30 Steven Karlish, Weizmann Institute, Dept. of Biological Chemistry,
Rhehovot, Israel Development of isoform selective cardiac glycosides as agents to reduce intraocular pressure 14.30-15.00 Jan Koenderink, Radboud University Medical Center, Nijmegen, Holland
Na+,K+-ATPase isoforms in disease
15.00-15.30 Rikke Holm, Aarhus University, Biomedicine, Physiology, Denmark
Rescue of Na+ affinity in aspartate-928 mutants of Na+,K+-ATPase by secondary mutation of glutamate-314 15.30-16.00 Coffee

16:00-16.30 Steven Clapcote, Faculty of Biological Sciences, Leeds University,
Leeds, UK Characterization of cognitive deficits in mice with an alternating hemiplegia of childhood-linked mutation
16.30-17.00 Thomas Holm, Aarhus University, Biomedicine, Biochemistry, Aarhus,
A mouse model for Alternating Hemiplegia in Childhood
17.00-17.30 Karin Lykke-Hartmann, Aarhus University, Biomedicine, Biochemistry,
Translational studies of sodium pump knock-in mouse models
17.30-18.00 Vladimir Matchkov, Aarhus University, Biomedicine, Physiology,
The α2 isoform of Na+,K+-ATPase modulates vascular tone via activation of Src kinase (Src) signaling pathway: a lesson from mouse model for Familial Hemiplegic Migraine Type 2 (FHM2) 18.00-19.00 Dinner

Evening free
22.00
Midnight snack (Natmad)
TUESDAY MAY 19TH

SESSION 2:

SENSING THROUGH CHANNELS

Coordinator: Helle Praetorius, Biomedicine, Aarhus, Denmark

9.00-9.30
Kate Poole, Department of Neuroscience, Max-Delbrück Center for
Molecular Medicine, Berlin, Germany
Tuning Piezo ion channels to detect molecular-scale movements relevant
for fine touch

9.30-10.00
Stuart Johnson, Department of Biomedical Science, University of
Sheffield, UK Calcium entry into stereocilia drives adaptation of the
mechanoelectrical transducer current of mammalian cochlear hair cells
10.00-10.30 Coffee

10.30-11.00 Sarah Falk, Department of Drug Design and Pharmacology, University of
Copenhagen, Denmark Pain without Nociceptors? Nav1.7-Independent Pain Mechanisms
11.00.11.30 Peter Zygmunt, Clinical Chemistry and Pharmacology, Department of
Laboratory Medicine, Lund University, Sweden Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain
11.30-12.00 Arend Vogt, Humboldt University zu Berlin, Biology, Germany
The diversity of light-driven proton pumps and their conversion into proton channels
12.00 - 13.00 Lunch
SESSION 3:
STRUCTURE AND FUNCTION OF MEMBRANE RECEPTORS

Coordinator: Christian Brix Folsted Andersen, Aarhus, Denmark

13.30-14.00 Kristian Stødkilde-Jørgensen, Aarhus University, Biomedicine,
Biochemistry, Denmark Hemoglobin Piracy by Trypanosome parasites 14.00-14.30 Jan Terje Andersen, Oslo University Hospital, Centre for Immune
Regulation and Depart. of Immunology, Norway The role of albumin receptors in regulation of albumin homeostasis: implications for drug delivery
14.30-15.00 Gregers Rom Andersen, Aarhus University, Department of Molecular
Biology and Genetics, Denmark The structure of the RAGE:S100A6 complex reveals a new mode of homodimerization for S100 proteins
15.00-15.30 Coffee

15.30-16.00 Mette Madsen, Aarhus University, Biomedicine, Biochemistry, Denmark
The membrane receptor megalin modulates proliferation and survival rates in melanoma cells – could it be a novel biomarker and therapeutic target in melanoma cancer?
16.00-16.30 Tina Storm, Aarhus University, Biomedicine, Anatomy Denmark
Megalin in ocular health and disease
16.30–17.00 Free time
17.00-18.00 Keynote speaker:

Prof. Christoph Korbmacher, Friedrich Alexander University Erlangen
Nürnberg, Germany
The epithelial Na+ channel ENaC


18.00-19.00 Dinner

SESSION 4:
Posters and wine:
19.00 -21.00
Poster contributions are listed alphabetically, by first author´s last name. 1. Renée Brekelmans, Aarhus University, Biomedicine, Physiology
Does hydochlorothiazide acidify the urine?
2. Henriette Christensen, Aarhus University, Biomedicine, Anatomy
Luminal acid/base transporting proteins in the choroid plexus epithelium 3. Steen Fagerberg, Aarhus University, Biomedicine, Physiology
P2X receptor desensitization by high concentrations of ATP changes the survival rate of THP-1 attacked by RTX toxins Hlya and LTxA. 4. Anne-Sofie Greve Christensen, Aarhus University, Biomedicine, Physiology
The effect of P2-receptor inhibition on the cytotoxic effects of α-haemolysin from E. coli – a murine sepsis model 5. Gitte Tindbæk Nielsen, Aarhus University, Biomedicine, Biochemistry
Is megalin involved in regulating the apoptotic/anti-apoptotic apparatus of melanoma cells? 6. Jette Skov Alstrøm, Copenhagen University, Institute for Cellular and Molecular
Isoform-specific phosphorylation-dependent regulation of connexin hemichannels 7. Annette Buur Steffensen, Copenhagen University, Institute for Cellular and
Molecular Medicine Cotransporter-mediated cerebrospinal fluid formation 8. Anna Thorsø Larsen, University of Copenhagen, Biology, Molecular Integrative
Physiology The purinergic P2X7receptor is involved in glucose-stimulated ATP release and ß-cell proliferation 9. Mai-Britt Thomsen, Aarhus University, Biomedicine, Biochemistry
Does the cytosolic adaptor protein Dab2 assist melanoma megalin during endocytosis? Please hang up your poster in the morning, and let it remain up all of Tuesday.

22.00-
Natmad (Traditional late-night open sandwiches snack)
WEDNESDAY MAY 20TH

SESSION 5:

FREE COMMUNICATIONS IN MEMBRANE TRANSPORT

Coordinators: Markus Bleich, Kiel, Germany and Ivana Novak, Copenhagen, Denmark

9.00-9.20
Brian Roland Larsen, University of Copenhagen, Institute for Cellular
and Molecular Medicine, Denmark
GIutamate-induced astrocytic [Na+]i elevation – a mechanism to increase
K+ clearance via the Na+/K+-ATPase?

9.20-9.40
Inga Christensen, Aarhus University, Biomedicine, Anatomy, Denmark
Choroid plexus epithelial cells are polarized normally - but contain
unusually located proteins


9.40-10.00
Annette Buur Steffensens, University of Copenhagen, Institute for Cellular
and Molecular Medicine, Denmark
Mechanisms underlying spreading depolarization-induced dendritic beading

10.00-10.20 Marco Tozzi, University of Copenhagen, Biology, Molecular Integrative
Physiology, Denmark Expression and Function of H+/K+-ATPases in Pancreatic Ductal Adenocarcinoma
10.20-10.40 Coffee
10.40-11.00 Åsa Jönsson, Aarhus University, Biomedicine, Pharmacology, Molecular
Integrative Physiology, Denmark Characterization and effect on phosphate transport of mutations in the human SLC34A2 gene in pulmonary alveolar microlithiasis
11.00-11.20 Silke Härteis, Friedrich Alexander University Erlangen Nürnberg,
Germany Prostate specific antigen (PSA)/Kallikrein related peptidase 3 (KLK3) regulates TRPV4 cation channel function by activating proteinase-activated receptor-2 (PAR2)
11.20-11.40 Mette Christensen, Aarhus University, Biomedicine, Physiology,
Denmark [Ca2+]i oscillations and Il-6 release induced by α-haemolysin from Escherichia coli require P2 receptor activation in renal epithelia 11.40-12.00 Casper K. Larsen, Aarhus University, Biomedicine, Physiology, Denmark
Reduced renal K+ excretion with compensatory hyperaldosteronism in KCa1.1 channel ß2-subunit KO mice
12.00…
Final remarks

12.10 - 13.00 Lunch
Departure

Sponsors
We are very grateful for generous financial support for this year's Sandbjerg
meeting from:
• Aarhus Universitets Forskningsfond (AUFF) • Aarhus University Graduate School of Health Sciences • MEMBRANES Abstracts Orals Monday the 18th May 2015

Development of isoform selective cardiac glycosides as agents to reduce
intraocular pressure
Steven J.D.Karlish
Weizmann Institute, Dept. of Biological Chemistry, Rhehovot, Israel
The ciliary epithelium in the eye consists of pigmented cells (PE), that express
the α1ß1 isoform of Na,K-ATPase, and non-pigmented (NPE) cells that express
mainly the α2ß3 isoform. In principle, a Na,K-ATPase inhibitor with selectivity for
the α2ß3 isoform that penetrates the cornea, could effectively reduce intra-ocular
pressure, with minimal systemic or local toxicity. We have recently synthesized a
series of perhydro-1-4-oxazepine derivatives of digoxin (by HIO4 oxidation of the
third digitoxose and reductive amination with various R-NH2 substituents) and
showed that several derivatives have significant selectivity for human α2ß1 over
α1ß1 isoform complexes (up to 8-fold). Furthermore, when applied topically, the most α2-selective derivatives effectively prevented or reversed pharmacologically raised intraocular pressure in rabbits (Katz etal J.Biol.Chem 289, 21153, 2014). A recent structure of Na,K-ATPase, with bound digoxin, shows the third digitoxose moiety in proximity to one residue in the ß1 subunit. As a test of the hypothesis that sugars of digoxin might interact with the ß subunit, we synthesized a new series of perhydro-1-4-oxazepine derivatives of digoxin with diverse substituents. Indeed several derivatives have enhanced selectivity for α2ß3 over α1ß1 (_33-fold in the most optimal case, respectively). When applied topically these derivatives potently reduce either pharmacologically raised or basal intraocular pressure in rabbits. The isoform-selective digoxin derivatives must effectively penetrate the cornea and inhibit α2ß3, so reducing aqueous humour production and intraocular pressure. The experiments demonstrate the central role of the NPE sodium pump in production of aqueous humour. The isoform selective digoxin derivatives may become interesting drug leads for treatment of ocular hypertension or glaucoma. Na+/K+-ATPase isoforms in disease
Jan B. Koenderink, Weigand KM, Swarts HGP
Radboud University Medical Center, Nijmegen, Holland
Sporadic and familial hemiplegic migraine type 2 are rare forms of hemiplegic
migraine caused by mutations in the Na,K-ATPase α2 gene. Mutations in
ATP1A3, the gene encoding the α3-subunit of Na,K-ATPase, are associated with
the neurodevelopmental disorder Alternating Hemiplegia of Childhood and
Rapid-onset Dystonia-Parkinsonism. Although knowledge about the affected
genes is paramount, the next step in understanding the mechanism-of-disease
of these mutations requires biochemical characterization of Na,K-ATPase
containing these mutations. We have studied the functional consequences of
many disease causing ATP1A2 and ATP1A3 mutations. We have looked at
ATPase activity, phosphorylation, and ouabain binding, using purified membrane
fractions from baculovirus-infected insect cells expressing the above-mentioned
mutant enzymes. The tested mutants can be divided into different groups that
might help understand how the variation in symptoms observed within patients is
explained at the molecular level.
Rescue of Na+ Affinity in Aspartate-928 Mutants of Na+,K+-ATPase by
Secondary Mutation of Glutamate-314
Rikke Holm, Einholm AP, Andersen JP and Vilsen B
Aarhus University, Biomedicine, Physiology, Denmark
The Na+,K+-ATPase binds Na+ at three transport sites denoted I, II, and III, of
which site III is Na+ specific and suggested to be the first occupied in the
cooperative binding process activating phosphorylation from ATP. Here we
demonstrate that the asparagine substitution of the aspartate associated with
site III found in patients with rapid-onset dystonia parkinsonism or alternating
hemiplegia of childhood causes a dramatic reduction of Na+ affinity in the α1-,
α2-, and α3-isoforms of Na+,K+-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na+ coordinating residues in site III. Remarkably, the Na+ affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na+ binding and does not affect the E1-E2 conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na+ affinity is likely intrinsic to the Na+ binding pocket, and the underlying mechanism could be a tightening of Na+ binding at Na+ site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na+,K+-pump function in intact cells. Rescue of Na+ affinity and Na+ and K+ transport by second-site mutation is unique in the history of Na+,K+-ATPase and points to new possibilities for treatment of neurological patients carrying Na+,K+-ATPase mutations. A mouse model for Alternating Hemiplegia in Childhood

Thomas Holm, Isaksen TJ, Glerup S, Füchtbauer EM, Bøttger P, Heuck A,
Nissen P, Lykke-Hartmann K
Aarhus University, Biomedicine, Biochemistry, Aarhus, Denmark
Specific mutations in the Na+/K+ ATPase α3 isoform cause the rare
neurodevelopmental disease, Alternating Hemiplegia of Childhood (AHC).
Children with AHC exhibit a wide range of neurological symptoms including
hemiplegia, dystonia, ataxia, nystagmus, hyperactivity, seizures and
developmental delays. Often, episodes are triggered by stressful events. We will
present some of our latest data from our AHC mouse model, including
pharmacological approaches to rescue several disease phenotypes.
Translational studies of sodium pump knock-in mouse models
Bøttger P, Glerup S, Gesslein B, Illarionova NB, Isaksen TJ, Heuck A, Clausen
BH, Füchtbauer EH, Gramsbergen JB, Gunnarson E, Aperia A, Lauritzen M,
Lambertsen KL, Nissen P, Karin Lykke-Hartmann
Aarhus University, Biomedicine, Biochemistry, Denmark
Migraine is a complex brain disorder, and understanding the complexity of this
prevalent disease could improve quality of life for millions of people. Migraine co-
morbidity involves depression, and anxiety. Familial Hemiplegic Migraine type 2
(FHM2) is a subtype of migraine with aura and co-morbidities like
epilepsy/seizures, cognitive impairments and psychiatric manifestations. FHM2
disease-mutations locate to the ATP1A2 gene encoding the astrocyte-located
α2-isoform of the sodium-potassium pump (α2Na+/K+-ATPase). We show that knock-in mice heterozygous for the FHM2-associated G301R-mutation (α2+/G301R) phenocopy FHM2 by mimicking migraine symptoms. In vitro studies showed impaired glutamate uptake in hippocampal mixed astrocyte-neuron cultures from α2G301R/G301R E17 embryonic mice, and moreover, induction of cortical spreading depression (CSD) resulted in reduced recovery in α2+/G301R mice. NMDA-type glutamate receptor antagonists or progestin-only treatment reverted specific α2+/G301R behavioral phenotypes, and our findings support that haploinsufficiency of the α2-isoform encoding gene impairs K+ clearance and glutamate uptake. This study demonstrate that psychiatric manifestations are part of the FHM2-pathology and link the α2Na+/K+-ATPase to the glutamate system, with the female sex hormone cycle exerting aggravating effects hereon. The ɑ2 Isoform of Na+,K+-ATPase Modulates Vascular Tone via Activation
of Src kinase (Src) Signaling Pathway: a Lesson from Mouse Model for
Familial Hemiplegic Migraine Type 2 (FHM2)
Hangaard L, Staehr C, Bouzinova E, Lykke-Hartmann K, Xie Z, Sandow S,
Aalkjaer C, Vladimir Matchkov
Aarhus Univesity, Biomedicine, Physiology, Denmark
The vasogenic theory of migraine suggests that the aura is associated with
vasoconstriction-induced hypoxemia in the brain, while the subsequent
headache is caused by a rebound vasodilation. The alternative theory views
migraine as a neurologic disorder. Neither of the theories can alone account for
the pathophysiology of migraine.
Familial hemiplegic migraine type 2 (FHM2) has been shown associated with
point mutations in the a2 isoform of Na,K-ATPase, including G301R mutation.
The a2 isoform expresses and plays an important role in both neuronal and
vascular tissues, making it difficult to prioritize these tissues for the pathology of
migraine.
Homozygotes mice bearing G301R die very early but heterozygotes (+/-G301R)
are viable. Vascular functions of middle cerebral (MCA) and mesenteric small
arteries (MSA) from wild type (WT) and +/-G301R mice were compared. A
peptide inhibiting the Na,K-ATPase-dependent Src kinase activation, pNaKtide,
was used to analyze the potential role of this signaling pathway.
There was no difference in blood pressure (telemetry) between WT and +/-
G301R mice. MCA from +/-G301R mice showed a significant reduction in the a2
isoform expression (to 53±12%, n=5; whole-mount immunohistochemistry).
Accordingly, ouabain in the concentrations up to 3•10-5 M constricted MCA from
+/-G301R significantly less than WT. MCA diameter was larger in +/-G301R
compared with WT. MCA constricted stronger to U46619, endothelin and K+-
depolarization. This was associated with sensitization to [Ca2+]i. pNaKtide
significantly suppressed contraction and abolished differences between groups.
In contrast, there was no difference in the contractile responses for MSA from +/-
G301R and WT. Moreover, pNaKtide significantly suppressed the contraction
only of WT MSA.
Elevated [K+]out hyperpolarized and relaxed MCAs from +/-G301R mice more
than from WT. These responses were Ba2+ sensitive. Accordingly, arteries from
+/- G301R had elevated mRNA for inward rectifying K+ channels; 190±32% of
the WT.
FHM2-associated mutation leads to elevated contractility and relaxation to
metabolically elevated K+ of cerebral arteries. MCA hypercontractility is mediated
via Src activation. We suggest these changes are involved in the
vasoconstriction-induced hypoxemia in the brain during the aura and in the
rebound vasodilation during subsequent headache. This signaling is prominent
in cerebral but not in peripheral circulation.

Abstracts Orals Tuesday the 19th May 2015

Tuning Piezo ion channels to detect molecular-scale movements relevant
for fine touch
Wetzel C, Goek C, Herget R, Lapatsina L, Ngo HD, Lewin G, Kate Poole
Department of Neuroscience, Max-Delbrück Center for Molecular Medicine,
Berlin, Germany
In sensory neurons, mechano-transduction is sensitive, fast and requires
mechanosensitive ion channels. In order to quantitatively study this
mechanosensitive channel activity we have developed a new method to directly
monitor mechanotransduction at defined regions of the cell-substrate interface.
We have found that molecular-scale (approx. 13 nm) displacements are
sufficient to gate mechano-sensitive currents in mouse touch receptors. Using
neurons from knockout mice, we found that displacement thresholds increase by
one order of magnitude in the absence of the membrane scaffolding protein,
stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of
mammalian stretch-activated ion channels, and we have shown that STOML3,
but not other stomatin-domain proteins, reduces the activation threshold for
Piezo1 and Piezo2 currents in a heterologous system to approx 10 nm.
Structure–function experiments localize the Piezo modulatory activity of
STOML3 to the stomatin domain, and higher-order scaffolds are a prerequisite
for function. As such, we have shown that STOML3 is the first potent modulator
of Piezo channels that tunes the sensitivity of mechanically gated channels to
detect molecular-scale stimuli relevant for fine touch. The dependence of
STOML3 function on its oligomerisation allowed us to design and execute a
small molecule screen for compounds that alter STOML3 self-association; we
identified small molecules that, when exogenously applied, can modulate the
mechano-sensitivity of sensory neurons via changes in STOML3 scaffolds.
Calcium entry into stereocilia drives adaptation of the mechanoelectrical
transducer current of mammalian cochlear hair cells
Stuart L. Johnson, Corns LF, Marcotti W
Department of Biomedical Science, University of Sheffield, UK
Mammalian cochlear hair cells transduce sound waves into electrical signals
which are then relayed to the auditory centres of the brain. Sound waves
displace the stereociliary bundles which project from the apical surface of hair
cells. Mechanotransducer (MT) channels located at the tips of these stereocilia
are opened by the movement of the hair bundle towards the taller stereocilia. In
lower vertebrate hair cells, calcium entry through open MT channels causes
channel adaptation, resulting in a lower channel open probability and a resetting
of their operating range. This ensures that the hair cells always operate around
their maximum sensitivity. It remains uncertain whether calcium-dependent
adaptation is also present in mammalian cochlear hair cells.
We mechanically deflected the hair bundles of both outer and inner hair cells of
mice using a piezo-driven fluid jet. We found that when the calcium influx into
hair cell stereocilia was abolished/decreased by either depolarising the cells to
near the calcium reversal potential or exposing the cells to the in vivo
endolymphatic calcium concentration (40 microM), the adaptation of the MT
current was abolished. The resting open probability, which is dependent on the
degree of adaptation, was also increased under these conditions. Decreasing
the amount of free calcium within the cell by increasing the concentration of the
intracellular calcium buffer BAPTA also abolished all manifestations of
adaptation. These findings show that MT current adaptation in mouse auditory
hair cells is directly modulated by calcium.
Pain without nociceptors? Nav1.7-independent pain mechanisms
Minett MS, Sarah Falk, Santana-Varela S, Bogdanov YD, Nassar MA, Heegaard
AM, Wood JN
Department of Drug Design and Pharmacology, University of Copenhagen,
Denmark
Nav1.7, a peripheral neuron voltage-gated sodium channel, is essential for pain
and olfaction in mice and humans. We examined the role of Nav1.7 as well as
Nav1.3, Nav1.8, and Nav1.9 in different mouse models of chronic pain.
Constriction-injury-dependent neuropathic pain is abolished when Nav1.7 is
deleted in sensory neurons, unlike nerve-transection-related pain, which requires
the deletion of Nav1.7 in sensory and sympathetic neurons for pain relief.
Sympathetic sprouting that develops in parallel with nerve-transection pain
depends on the presence of Nav1.7 in sympathetic neurons. Mechanical and
cold allodynia required distinct sets of neurons and different repertoires of
sodium channels depending on the nerve injury model. Surprisingly, pain
induced by the chemotherapeutic agent oxaliplatin and cancer-induced bone
pain do not require the presence of Nav1.7 sodium channels or Nav1.8-positive
nociceptors. Thus, similar pain phenotypes arise through distinct cellular and
molecular mechanisms. Therefore, rational analgesic drug therapy requires
patient stratification in terms of mechanisms and not just phenotype.
Human TRPA1 is intrinsically cold- and chemosensitive with and without
its N-terminal ankyrin repeat domain
Moparthia L, Survery S, Kreir M, Simonsen C, Kjellbom P, Högestätt ED,
Johanson U and Peter M. Zygmunt
Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund
University, Sweden
The discovery of transient receptor potential (TRP) ion channels, including the
TRP subtype A1 (TRPA1), as molecular chemo- and thermosensors has
opened-up new avenues for understanding how organisms monitor the
physicochemical environment.
As discovered by us, the mammalian TRPA1 is activated by plant-derived thiol-
reactive electrophilic compounds and oxidants, including isothiocyanates and
diallyl disulfide from mustard and garlic. This original finding and those of
numerous subsequent studies have consolidated mammalian TRPA1 as a prime
detector of tissue damaging environmental chemicals and pro-inflammatory
compounds. Several TRPA1 homologues exist in the animal kingdom, and the
ability of TRPA1 to sense potentially harmful electrophilic compounds has been
conserved for 500 millions of years, whereas the thermosensitive properties of
TRPA1 have diverged later.
As thermosensors, mammalian and insect TRPA1 look like yin yang. Rat and
mouse TRPA1 has been suggested to respond to cold temperatures below
20°C, whereas fruit fly (Drosophila melanogaster) and mosquito (Anopheles
gambiae) TRPA1 seem to respond to warm temperatures above 25°C. Ever
since the identification of mouse, rat and human TRPA1 in sensory neurons, a
role for TRPA1 as a noxious cold sensor has been controversial. Here. we have
cloned and purified TRPA1 from Homo sapiens and the malaria mosquito
Anopheles gambiae and provide evidence that these TRPA1 ion channels are
indeed intrinsically cold and heat sensitive proteins, respectively.
Chemical control of TRPA1 activity may provide novel drug treatments of pain
and strategies for developing insect and arachnid repellents for prevention of
human diseases, such as malaria and tick-born encephalitis.
The diversity of light-driven proton pumps and their conversion into proton
channels
Arend Vogt, Hegemann P
Humboldt University zu Berlin, Biology, Germany
Microbial rhodopsins are integral seven-transmembrane helix proteins which
bind covalently all-trans-retinal as light sensitive chromophore. They are
subdivided into sensory rhodopsins, ion channels and ion pumps. Light-driven
ion pumps transport protons, sodium or chloride across the plasma membrane
against their electrochemical gradient. Bacteriorhodopsin (BR) from
Halobacterium salinarum is the most notable proton pump and transports
protons out of the cell. More recently proton pumps have been employed in
neuroscience as optogenetical tools for silencing of neuronal activity by
hyperpolarization or as voltage sensors.
It was generally assumed that all light-driven microbial proton pumps behave
basically in the same matter like bacteriorhodopsin. We analyzed a variety of
proton pumps using two-microelectrode voltage-clamp measurements (TEVC) of
Xenopus leavis oocytes. We have found that the naturally occurring proton
pumps show different behaviors at high electrochemical load, i.e. low
extracellular pH and negative voltage. Photocurrents of Bacteriorhodopsin and
the rhodopsin from the eukaryotic microalga Coccomyxa subellipsoidea (CsR)
are always outward directed and inactivate at high load. In contrast, the
rhodopsins from Exiguobacterium sibiricum (ESR) and from the cyanobacterium
Gloeobacter violaceus (GR) show inward directed photocurrents at high load.
The rhodopsin Arch3 from the archaeon Halorubrum sodomense is well
established as optogentical tool and shows weak inward directed photocurrents
at high load.
We have used CsR for an efficient mutagenesis study and identified key
determinants for the directivity and the power of the pumps. Mutations at position
R83 and Y57 (BR numbering) converted CsR into an operational proton channel
with inward or outward rectification depending on the replacement. Such light-
activated proton pumps will be of great interest for optogenetic applications in
which a specific proton release is required (e.g., in lysozymes or neuronal
vesicles).
Hemoglobin Piracy by Trypanosome parasites
Kristian Stødkilde-Jørgensen, Andersen CBF
Aarhus University, Biomedicine, Biochemistry, Denmark
Human African Trypanosomiasis, also known as sleeping sickness, is a disease
caused by protozoan parasites of the Trypanosoma genus. The drugs available
for treating an infection are all associated with severe adverse effects and thus
new ways to treat the disease are desperately needed. In this regard,
understanding how the parasites take up exogenous ligands is interesting. The
parasites cannot synthesize heme themselves, yet require this prosthetic group
for incorporation into hemoproteins. The need for exogenous heme is
accommodated by a haptoglobin-hemoglobin receptor (HpHbR). This receptor
enables uptake of hemoglobin (Hb) bound by the plasma protein haptoglobin
(Hp). Interestingly, HpHbR is essential for human immunity against all but two
trypanosome subspecies. The immunity arises as the parasites are tricked into
taking up a trypanolytic compound that mimics the Hp-Hb com-plex.
To understand the underlying mechanisms of Hb uptake and its role in human
innate immunity, we crystallized and obtained the structure of HpHbR while
binding the Hp-Hb complex. In the present talk, I will show how the structure not
only answers key questions regarding Hb uptake, but how it also showcases an
evolutionary arms race between humans and trypanosomes.
The role of albumin receptors in regulation of albumin homeostasis:
implications for drug delivery
Jan Terje Andersen
Oslo University Hospital, Centre for Immune Regulation and Department of
Immunology, Norway
Albumin is the most abundant protein in blood and acts as a molecular taxi for a
plethora of small insoluble substances such as nutrients, hormones, metals and
toxins. In addition, it binds a range of medical drugs. It has an unusually long
serum half-life of almost 3 weeks, and although the structure and function of
albumin has been studied for decades, a biological explanation for the long half-
life has been lacking. Now, recent research has unravelled that albumin-binding
cellular receptors play key roles in homeostatic regulation of albumin. Here, I will
discuss our current understanding of albumin homeostasis with a particular focus
on the impact of the cellular receptors, namely the neonatal Fc receptor (FcRn)
and the cubilin-megalin complex, and their importance on uses of albumin in
drug delivery.
The structure of the RAGE: S100A6 complex reveals a new mode of
homodimerization for S100 proteins
Yatime L1, Betzer C2, Mortensen S1, Jensen PH2 and Gregers Rom Andersen1
1. Department of Molecular Biology and Genetics, Aarhus University, Denmark.
2. Danish Research Institute of Translational Neuroscience - DANDRITE,
Department of Biomedicine, Aarhus University, Denmark. S100 proteins constitute a large family of Ca2+-dependent regulators of homeostatic processes. Besides functioning intracellularly in calcium homeostasis, cell growth and differentiation, cytoskeleton dynamics, and energy metabolism, S100 proteins can be relocalized to the extracellular compartment where they act as damage-associated molecular patterns by becoming ligands of the receptor for advanced glycation end-products (RAGE), a pattern recognition receptor sensing endogenous stress signals associated with inflammation. Many S100 proteins are overexpressed in tumors and, through their signaling network, facilitate the communication between cancer and stromal cells, thereby maintaining an inflammatory microenvironment favorable to tumor growth and metastasis. Despite the importance of RAGE-S100 crosstalk in sustaining inflammation, the structural basis for S100 proteins interaction and signal transduction through RAGE is unknown. To gain insights into S100 proteins recognition mode by RAGE, we have undertaken the biochemical and structural characterization of various RAGE:S100 complexes, using analytical size exclusion chromatography and X-ray crystallography. The structure of the isolated human RAGE ectodomain was determined and revealed that the unliganded receptor is prone to homodimerization via its highly basic V domain. We also managed to obtain the first crystallographic structures of a RAGE:S100 complex between the human RAGE ectodomain and both human and mouse S100A6. The structures revealed a binding site for S100A6 located primarily in the RAGE C1 domain, thus being quite different from previous propositions, and suggested that zinc can further stabilize the RAGE:S100A6 interaction. Cell-based assays confirmed that this binding mode also occurs with the endogenous, cell-bound receptor. Furthermore, the structures revealed that S100A6 binding induces a novel dimeric conformation of RAGE that appears suited for signal transduction and intracellular effector recruitment. Surprisingly, the S100A6 ligand adopts a Zn2+-stabilized dimeric conformation radically different from known S100 dimer structures. Sequence analysis and modeling suggest that this conformation may be adopted by a variety of S100 proteins in the presence of oxidative conditions and/or extracellular divalent cations. These results provide valuable insights on S100 ligand recognition mode by RAGE and have implications for the targeting of S100 proteins in anti-cancer therapies. The membrane receptor megalin modulates proliferation and survival rates
in melanoma cells – could it be a novel biomarker and therapeutic target in
melanoma cancer?
Andersen RK1, Hammer K1, Hager H2, ChristensenJN1, Ludvigsen M1, Honoré
B1, Thomsen MBH1 and Mette Madsen1
1. Department of Biomedicine, Aarhus University, Denmark
2. Department of Pathology, Aarhus University Hospital, Denmark.
If melanoma cancer is detected at an early stage, where it is still localized to the
skin, the patient holds a good prognosis. Patients with early stage melanoma
cancer are usually cured by surgical resection of the melanocytic lesion.
However, if melanoma cancer spreads beyond the regional lymph node, the
prognosis is poor and only about 10 % of the patients diagnosed with metastatic
melanoma with distant metastases survive. Unfortunately, no biomarkers have
been established than can identify the most aggressive primary melanoma
tumors, predict metastasation, and point towards the need of adjuvant treatment.
Aggressive melanoma cells are characterized by increased proliferative activity,
improved anti-apoptotic machinery, as well as enhanced metastatic potential.
We recently hypothesized that acquired expression of megalin by melanocytic
lesions/melanoma tumors improves cancerous characteristics. Megalin is an
endocytic receptor known to bind an extensive number of different ligands.
Megalin either mediates their uptake from the extracellular environment or
facilitates their intracellular trafficking. Megalin has been widely studied in
relation to embryonic development; especially of the brain, where it is known to
play a fundamental role in modulating sonic hedgehog signaling events.
Our initial study has revealed for the first time that megalin is frequently
expressed in melanomas and melanoma metastases. Megalin is only rarely
expressed in benign nevi. Our functional analyses have indicated that melanoma
megalin associates with the endocytic apparatus and that it can be internalized
from the cell surface to intracellular vesicles. Groundbreaking, our results
indicate that sustained megalin expression in melanoma cells is crucial for cell
maintenance. We observed that siRNA-mediated reduction of melanoma cell
expression of megalin significantly decreased melanoma cell proliferation and in
particular survival rates.
Our study has thus established a platform for acknowledging megalin as a
potential new biomarker of aggressive melanoma cells. It might furthermore be
addressed as a future therapeutic cancer target, specifically in melanoma.
Megalin in ocular health and disease
Tina Storm1, Heegaard S2,3, Christensen EI1, Nielsen R1
1. Department of Biomedicine, Aarhus University, Denmark
2. Eye Pathology Institute, Department of Neuroscience and Pharmacology,
University of Copenhagen, Denmark 3. Department of Ophthalmology, Glostrup Hospital, University of Copenhagen, Mutation of the megalin-encoding gene (LRP2) causes the rare Donnai-Barrow/ Facio-Oculo-Acoustico-Renal Syndrome which is partially characterized by high-grade myopia. Previous studies of renal megalin function have established that megalin is crucial for conservation of renal filtered nutrients and plasma proteins but the role of megalin in ocular physiology and pathology is unknown. To elucidate this, we have investigated ocular megalin expression and the ocular phenotype of megalin-deficient mice. Topographical and subcellular localization of megalin as well as the ocular phenotype of megalin-deficient mice were examined with immunological techniques using light-, confocal-, and electron microscopy. In normal mice, megalin was identified in vesicular structures in the retinal pigment epithelium (RPE) and non-pigmented ciliary body epithelium (NPCBE). Histological investigations of ocular mouse tissue from megalin-deficient mice revealed a severe high myopia phenotype with grossly enlarged RPE melanosomes as well as abnormal ciliary body development. In conclusion, the complex and severe ocular phenotype observed in the megalin-deficient mice suggests that developmental abnormalities may play a key role in the the high myopia observed in all Donnai-Barrow Syndrome patients and that megalin harbors important roles in ocular development and/or physiology. Finally, our data shows that megalin-deficient mice may provide an invaluable model for future studies of megalin in ocular physiology and pathology. The epithelial sodium channel (ENaC)
Christoph Korbmacher
Friedrich Alexander University Erlangen-Nürnberg, Germany
The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin
family of ion channels. ENaC is localized in the apical membrane of epithelial
cells and is the rate limiting step for sodium absorption in epithelial tissues
including the aldosterone-sensitive distal nephron (ASDN) and respiratory
epithelia. Abnormal ENaC activation in the ASDN may cause sodium retention
and arterial hypertension. ENaC regulation in the ASDN is highly complex with
aldosterone-dependent and independent mechanisms. ENaC activity can be
stimulated by norepinephrine which may contribute to the hypertensive effect of
increased renal sympathetic activity. A unique feature of ENaC is its proteolytic
activation which involves specific cleavage sites and the release of inhibitory
peptide fragments. However, the physiologically relevant proteases involved in
ENaC regulation remain to be identified. In nephrotic syndrome filtered
plasminogen is converted to plasmin by tubular urokinase. ENaC activation by
tubular plasmin may contribute to sodium retention in nephrotic syndrome. ENaC
mutations identified in patients with atypical cystic fibrosis (CF) mimic proteolytic
channel activation and may play a role in the pathophysiology of pulmonary
symptoms in CF. Interestingly these latter mutations do not seem to cause
arterial hypertension unlike the gain-of-function mutations identified in patients
with Liddle's syndrome (pseudohyperaldosteronism).
Abstracts Orals Wednesday 20th May 2015

GIutamate-induced astrocytic [Na+]i elevation – a mechanism to increase
K+ clearance via the Na+/K+-ATPase?
Brian Roland Larsen, Holm R, Vilsen B and MacAulay N
University of Copenhagen, Institute for Cellular and Molecular Medicine,
Denmark
Neuronal activity in the brain is associated with a transient increase in the
extracellular K+ concentration. Accumulated K+ would result in a depolarization of
neurons and glia and disturbance of neuronal signaling. The excess K+ is initially
cleared by surrounding astrocytes by mechanisms involving the Na+/K+-ATPase.
During the majority of neuronal activity, glutamate increases in the synaptic cleft
along with K+. Glutamate is swiftly re-absorbed by astrocytic Na+-coupled
glutamate transporters, thereby elevating the intracellular Na+ concentration. The
Na+/K+-ATPase consists of an α- and a ß-subunit, with several isoforms of each
subunit present in the mammalian brain. The combination of isoforms control the
functional characteristics of the Na+/K+-ATPase, e.g. its response to changes in
extracellular K+ or intracellular Na+. Though the astrocytic α2ß2 isoform
constellation responds directly to extracellular K+ above basal levels, it has been
suggested that the intracellular astrocytic Na+ concentration may govern Na+/K+-
ATPase activity and consequently control its ability to clear K+ from the
extracellular space. It thus remains unresolved whether the Na+/K+-ATPase-
mediated clearance of K+ is driven by the elevation of [K+]o or [Na+]i. The
apparent intracellular Na+ affinity of isoform constellations involving ß2 has
remained elusive due to inherent expression of ß1 in most expression systems
as well as the technical challenges in measuring an intracellular affinity in an
intact system. We therefore expressed the different astrocytic isoform
constellations in Xenopus oocytes (α1ß1, α2ß1, α1ß2, α2ß2) and determined the
apparent Na+ affinity by two different methods: An enzymatic phosphorylation
assay on harvested membranes and two-electrode voltage clamp on intact cells
gradually loaded with Na+. The obtained Na+ affinities indicated that the Na+/K+-
ATPase was near saturation at basal astrocytic [Na+]i, irrespective of isoform
constellation, and was not stimulated by parallel glutamate transporter activity.
Ongoing extracellular hippocampal slice recordings of stimulus-induced [K+]
transients, using ion-sensitive microelectrodes, will reveal whether a glutamate
transporter-induced increase in [Na+]i contributes to Na+/K+-ATPase-mediated K+
clearance in a setting approximating native conditions.
Choroid plexus epithelial cells are polarized normally - but contain
unusually located proteins
Inga B Christensen, Damkier HH and Praetorius J
Aarhus University, Biomedicine, Anatomy, Denmark
The choroid plexus epithelium secretes the majority of the cerebrospinal fluid.
This important brain-supporting function is enabled by the composition of
transporter proteins in the plasma membrane of the epithelial cells. The choroid
plexus cells are unique in that certain transporter proteins are located in different
cellular domains when compared to most other polarized epithelial cells. One
example is the ubiquitously expressed Na+,K+-ATPase, which is found in the
luminal membrane domain of choroid plexus cells - opposite its typical
basolateral localization. The polarity of epithelial cells is established on the basis
of extracellular clues. Polarity proteins inside the cells translate these clues into
a specific orientation, and this determines the organization of all intracellular
components. From vast knowledge of protein trafficking, it is believed that a
given transporter protein is always inserted into a specific cellular domain of a
polarized epithelial cell. The deviations from this in the choroid plexus epithelium
continue an important cell biological question; what determines the cellular
localization of transporter proteins?
As a first step to elucidate this question, thus clarify the mechanism behind
atypical localization of certain proteins in the choroid plexus cells, we utilized
immunohistochemistry. By this approach, we wanted to clarify whether
deviations in the localizations of polarity proteins form the basis for the unusual
localization of membrane proteins in the choroid plexus cells. So far, the
immunohistochemical stainings of choroid plexus cells showed three different
polarity proteins, PAR-1, PAR-3 and aPKC, located in each their cellular domain.
PAR-1 is found near the basal plasma membrane, PAR-3 at the tight junctions,
and aPKC beneath the luminal membrane. All three were located as expected
for normally polarized cells. We conclude that these central polarity proteins are
distributed normally in the choroid plexus cells, and that they do not seem to be
involved in the unusual localizations of key transport proteins in these cells. More
work in the research topic is required to clarify what enables unusual localization
of certain transporter proteins in the otherwise normal choroid plexus cells, and
furthermore to answer what determines the localization of transporter proteins in
all epithelial cell types.
Mechanisms underlying spreading depolarization-induced dendritic
beading
Annette B. Steffensen, Damkier HH, Tritsaris K, Prætorius J, and MacAulay N
University of Copenhagen, Institute for Cellular and Molecular Medicine,
Denmark
Spreading depolarizations (SDs) are waves of sustained neuronal and glial
depolarization that propagate massive disruptions of ion gradients through the
brain. SD is associated with migraine aura and recently recognized as a novel
mechanism of injury in stroke and brain trauma patients. SD leads to neuronal
swelling as assessed in real time with 2-photon laser scanning microscopy
(2PLSM). Pyramidal neurons do not express aquaporins and thus display low
inherent water permeability. Yet, SD rapidly induces focal swelling (beading)
along the dendritic shaft by unidentified molecular mechanisms. To address this
issue, we induced SD in hippocampal slices by focal KCl-microinjection and
visualized the ensuing dendritic beading by 2PLSM.
We confirmed that dendritic beading failed to arise during large (100 mOsm)
hyposmotic challenges, underscoring that neuronal swelling does not occur as a
simple osmotic event. Dendritic beading was strictly dependent on the presence
of Cl- and accordingly, combined blockade of Cl--coupled transporters, in
addition to lactate transporters, led to a significant reduction in dendritic beading
without interfering with SD. Furthermore, our in vivo data showed a strong
inhibition of dendritic beading upon pharmacological blockage of these
cotransporters. We propose that SD-induced dendritic beading takes place as a
consequence of the altered driving forces and thus activity for these
cotransporters, which by transport of water during their translocation mechanism
may generate dendritic beading independently of osmotic forces.
Expression and Function of H+/K+-ATPases in Pancreatic Ductal
Adenocarcinoma

Marco Tozzi, Giannuzzo A, Novak I
University of Copenhagen, Biology, Molecular Integrative Physiology, Denmark
Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the most
lethal malignancies, with median survival of less than one year and overall 5-
year survival less than 5% (1). Potential targets of cancer therapy are ion
channels and transporters involved in acid/base transports which are
deregulated in cancers and contribute to create a reversed pH gradient
(intracellular pH higher than extracellular). This favors cellular proliferation,
invasion, metastasis and resistance to chemotherapy in in a variety of cancers
(3) and this can be true also for PDAC. Recent studies have shown that
pancreatic ducts express gastric and non-gastric H+/K+-ATPases and they have
a physiological role in pH regulation and bicarbonate secretion (2).
The aim of the present study was to investigate whether these proton pumps are
expressed and functional in PDAC cell lines and whether proton pump inhibitors
(PPIs) can affect cancer progression.
Data from western blot quantification analysis and quantitative PCR revealed
different expression of the gastric HKα1, non-gastric HKα2 and HKß subunits
(ATP4A; ATP12A; ATP4B) of H+/K+-ATPases in the pancreatic cancer cell lines
compared with the normal one. The protein expression was also verified with
immunocytochemical analyses. In BxPC-3, Capan-1 and HPDE cells
omeprazole and SCH28080 inhibited cell proliferation up to 50% in a dose-
dependent manner and the migration rate up to 70%, particularly on cancer cells.
In conclusion, these data provide for the first time evidence that the H+/K+-
ATPases are involved in PDAC progression and that PPIs can attenuate
proliferation and migration of pancreatic cancer cells. This could suggest a
possible use of PPIs in cancer therapy.
Characterization and effect on phosphate transport of mutations in the
human Slc34a2 gene in pulmonary alveolar microlithiasis
Åsa Lina Jönsson1, Hernando N3, Hilberg O2, Bendstrup E2, Christensen JH1,
Mogensen S1, Wagner C3, Simonsen U1
1. Department of Biomedicine, University of Aarhus, Denmark
2. Department of Respiratory Medicine and Allergology, Aarhus University
Hospital, Denmark
3. Institute of Physiology, University of Zurich
The sodium phosphate co-transporter (NaPi-IIb) is a member of the sodium-
phosphate co-transporter family SLC34A. These co-transporters are expressed
in several tissues and play a major role in the homeostasis of inorganic
phosphate. Mutations in the SLC34A2 gene, that encodes NaPi-IIb, cause defect
cell-uptake of phosphate, which leads to formation of calcium-phosphate
concretions in the lungs, as seen in pulmonary alveolar microlithiasis (PAM).
PAM is a very rare disease with less than 600 patients reported worldwide. The
clinical course varies, from asymptomatic to severe disease with progression into
lung fibrosis, respiratory failure, and cor pulmonale. There is no known effective
treatment of PAM, with the exception of lung transplantation. We have earlier
described a new mutation in the SLC34A2 gene in two patients with PAM from
Aarhus University Hospital (1).
cDNA, encoding the wild type human NaPi-IIb and four mutants, were subcloned
into a vector optimized for expression in Xenopus oocytes. Mutations were
introduced by site-directed mutagenesis, the plasmids were linearized with NotI
and cRNA was synthesized using a T3 Message Machine kit. Oocytes from X.
laevis frogs were injected with the cRNA and subsequently radioisotope uptake
assays were performed. In addition, direct sequencing of SLC34A2 was
performed on blood samples from three PAM patients.
Preliminary results: The wild type SLC34A2 transported phosphate in the
presence of sodium, whereas the mutants showed similar uptake levels as the
non-injected oocytes. In addition, the expression levels of the wild type and the
mutants, investigated by Western Blot, showed the expected molecular weights.
Furthermore, three new mutations were found in three patients with PAM.
These preliminary findings show, as expected, a reduced phosphate uptake in
four mutants compared to the wild type SLC34A2. In addition, three novel
mutations were found in three patients with PAM.
This project will provide important new knowledge about the function of different
mutations in the SLC34A2 gene. The only currently existing treatment of the
PAM disease is lung transplantation. Hopefully, these findings may help to
develop a specific pharmacological treatment for the disease.
Prostate specific antigen (PSA)/Kallikrein related peptidase 3 (KLK3)
regulates TRPV4 cation channel function by activating proteinase-
activated receptor-2 (PAR2)
Silke Haerteis1, Sostegni S.1, Mihara K.3, Stenman U.-H.2, Koistinen H.2,
Hollenberg M.D.3, Korbmacher C.1.
1. Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Institut für Zelluläre
und Molekulare Physiologie, Erlangen, Germany
2. University of Helsinki and Helsinki University Central Hospital, Department of
Clinical Chemistry, Helsinki, Finnland
3. University of Calgary, Department of Physiology & Pharmacology, and
Department of Medicine, Calgary, Canada
Transient receptor potential vanilloid 4 (TRPV4), a member of the TRP channel
superfamily, is a non-selective cation channel. It has a broad spectrum of
physiological functions and is expressed in a wide range of tissues including the
prostate. However, the functional significance of TRPV4 in the prostate is still
unclear. KLK3 (kallikrein-related peptidase 3), also known as PSA (prostate-
specific antigen), is a member of the family of kallikrein-related peptidases and
has been linked to cancer-associated pathophysiology in particular in the
prostate. Although the proteinase targets for PSA in the prostate are not
established, other members of the KLK family of enzymes can signal by cleaving
and activating proteinase-activated receptors (PARs), including PAR2. Of note,
proteases can regulate TRPV4 via activating proteinase-activated receptor-2
(PAR2). Therefore, the aim of this study was to investigate whether PSA 1) is as
a potential regulatory protease of TRPV4 affecting its activity and 2) cleaves
PARs.
We determined TRPV4 activity by measuring whole-cell currents elicited by the
selective TRPV4 agonist GSK1016790A in Xenopus laevis oocytes
heterologously expressing human TRPV4 using the two-electrode voltage-clamp
technique. Active PSA was purified from pooled human seminal fluid by
immunoaffinity chromatography followed by anion exchange chromatography. To
test the effect of proteases on TRPV4 channel activity, oocytes were pre-
incubated for 30 min in the presence or absence of PSA. To characterize the
effect of PSA we used the PSA-inhibiting antibody or two PSA-stimulating
peptides.
Our data demonstrate that by activating PAR2, PSA can regulate the activity of
TRPV4 by showing a stimulatory effect on GSK1016790A-induced TRPV4
currents of four- to fivefold. The stimulatory effect of PSA on TRPV4 currents
was enhanced by PSA-stimulating peptides and was prevented by the PSA-
inhibiting antibody. Interestingly, the increase of TRPV4 currents by PSA was
essentially abolished by two inhibitors of protein kinase C (Go6983, GF109203X)
and by pre-incubation with calcium chelator BAPTA-AM indicating that the
stimulatory effect involves calcium signaling (Gq-coupled signaling pathway).
In conclusion, our work reveals a novel action of PSA in conjunction with PAR2
that may be relevant for prostate physiology and pathophysiology.
[Ca2+]i oscillations and Il-6 release induced by alpha-haemolysin from
Escherichia coli require P2 receptor activation in renal epithelia
Mette G. Christensen, Fagerberg SK, de Bruijn PI, Bjaelde RG, Jakobsen H,
Leipziger J, Skals M and Praetorius HA
Aarhus University, Biomedicine, Physiology, Denmark
Urinary tract infections are commonly caused by a-hemolysin (HlyA)-producing
Escherichia coli. In erythrocytes, the cytotoxic effect of HlyA is strongly amplified
by P2X receptors, which are activated by extracellular ATP, released from the
cytosol directly through the HlyA pore. In renal epithelia, HlyA causes reversible
[Ca2+]i oscillations, which trigger interleukin-6 (Il-6) and Il-8 release. We
speculate that this effect is caused by HlyA-induced ATP release from the
epithelial cells and successive P2 receptor activation.
Here, we demonstrate that HlyA-induced [Ca2+]i oscillations in renal epithelia
were completely prevented by scavenging extracellular ATP. In accordance,
HlyA was unable to inflict any [Ca2+]i oscillations in 132-1N1 cells, which lack
P2R completely. After transfecting these cells with the hP2Y2 receptor, HlyA
readily triggered [Ca2+]i oscillations, which were abolished by P2 receptor
antagonists. Moreover, HlyA-induced [Ca2+]i oscillations were markedly reduced
in medullary thick ascending limbs isolated from P2Y2 receptor deficient mice
compared to wild type. Interestingly, the following HlyA-induced Il-6 release was
absent in P2Y2 receptor deficient mice. This suggest that HlyA induces ATP
release from renal epithelia, which via P2Y2 receptors is the main mediator of
HlyA-induced [Ca2+]i oscillations and Il-6 release. This supports the notion that
ATP-signaling occurs early during bacterial infection and is key player in the
further inflammatory response.
Reduced renal K+ excretion with compensatory hyperaldosteronism in
KCa1.1 channel ß2-subunit KO mice

Casper K. Larsen, Sørensen MV, Praetorius HA, Leipziger J
Aarhus University, Biomedicine, Physiology, Denmark
The kidney is the primary organ responsible for excreting K+, ensuring whole
body K+ homeostasis by precisely matching K+ excretion to dietary K+ intake. K+
is secreted into the urine in the collecting ducts, and two distinct mechanisms for
K+ secretion exist; a constitutive mechanism mediated by ROMK (Kir1.1) in
principal cells and a flow-induced mechanism mediated by BK channels
(KCa1.1) in intercalated cells. Both mechanisms are up-regulated by
aldosterone. Here we studied renal K+ excretion in KO mice for the ß2-subunit of
the BK channel.
The ß2 KO mice have increased plasma aldosterone, low renin expression and
normal plasma [K+]. The low renin in ß2 KO mice indicates that
hyperaldosteronism was triggered by a K+ handling deficiency, rather than
hypotension and activation of the renin-angiotensin-aldosterone system. We
hypothesize that ß2 KO mice have decreased BK channel-mediated renal K+
secretion, which is compensated by hyperaldosteronism and up-regulation of
ROMK-mediated K+ secretion, allowing ß2 KO mice to maintain normal plasma
[K+]. In fact, when treated with eplerenone (mineralocorticoid receptor
antagonist) for 4 days, ß2 KO mice develop hyperkalemia (4.15 mM ± 0.13 in
WT vs. 4.60 mM ± 0.10 in KO, P = 0.013).
Urinary K+ excretion following oral K+ load (15% of normal daily intake) was not
different between WT and KO mice under control conditions. However, when
treated with eplerenone, ß2 KO mice had a significantly lower urinary K+
excretion rate (P=0.044) and significantly higher plasma [K+] 3 hours after oral K+
load (10.0 mM ± 0.4 in WT vs. 11.4 mM ± 0.5 in KO, P = 0.044). Our data
support that hyperaldosteronism in ß2 KO mice is part of a chronic
compensation to a decreased BK channel-mediated renal K+ secretion.

Isoform-specific phosphorylation-dependent regulation of connexin
hemichannels
Jette Skov Alstrøm, Hansen DB, Nielsen MS and MacAulay N
University of Copenhagen, Denmark
Connexins (Cx) form gap junction channels which are made up of two
connexons (hemichannels) expressed in the cell membrane of adjacent cells.
Unopposed hemichannels may open towards the extracellular matrix upon
stimulation by e.g. removal of divalent cations from the extracellular solution and
allow transmembrane flux of isoform-specific fluorescent dyes and
physiologically relevant molecules, such as ATP, and current (ions). Cx43 and
Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43
in their gap junction configuration and may also act to keep Cx43 hemichannels
closed. In contrast, the regulation of Cx30 hemichannels by PKC is unexplored.
To determine phosphorylation-dependent regulation of Cx30 and Cx43
hemichannels, these were heterologously expressed in Xenopus laevis oocytes
and their opening was induced with divalent cation free solution (DCFS).
Connexin dephosphorylation was promoted with PKC inhibitors which did not
affect hemichannel opening of either connexin. PKC activation had no effect on
Cx43-mediated hemichannel activity whereas both dye uptake and current
through Cx30 hemichannels were reduced. We detected no PKC-induced Cx30
or Cx43 internalization from the plasma membrane indicating that reduced Cx30
hemichannel activity occurred via a PKC-dependent gating event closing the
channel. In an attempt to resolve the PKC phosphorylation site on Cx30, alanine
mutations of seven putative cytoplasmic PKC consensus sites were created to
prevent phosphorylation (T5A, T8A, T102, S222A, S225A, S239A, and S258A).
All seven Cx30 mutations responded to PKC activation suggesting that Cx30, in
its hemichannel configuration, is not regulated by phosphorylation of a single
site. In conclusion, Cx30 – but not Cx43 – hemichannels close upon PKC
activation, illustrating that connexin hemichannels display not only isoform-
specific permeability profiles but also isoform-specific phosphorylation-
dependent regulation.

Does hydrochlorothiazide acidify urine?
Renée Brekelmans, de Bruijn P, Leipziger J
Aarhus University, Biomedicine, Denmark
The loop-diuretic furosemide inhibits NaCl reabsorption in the thick ascending
limb of Henle's loop (TAL) in the kidney, by blocking the Na+ Cl- K+
cotransporter, NKCC2. In addition to its diuretic effect, furosemide also provokes
an acute and pronounced acidification of the urine. This is traditionally explained
by an increased Na+ delivery to the connecting tubule (CNT) and cortical
collecting duct (CCD), which is believed to stimulate H+ secretion via the apical
H+-ATPase in a-intercalated cells. However, we have recently demonstrated that
the TAL itself plays an important role in the acidifying effect of loop diuretics by
activation of the apical Na+/H+ exchanger 3 (NHE3).
The reabsorption of NaCl in the distal convoluted tubule (DCT), located
upstream of the CNT/CCD, can be inhibited with thiazide diuretics. Similarly to
furosemide, thiazides also increase the delivery of Na+ to the CNT/CCD, but an
acute effect on urine acidification has to our knowledge never been reported.
This study intends to investigate the acute effect of NCC inhibition with
hydrochlorothiazide on urine pH and electrolytes in mice in an in vivo
experimental setup. Preliminary data suggests that hydrochlorothiazide does not
acidify the urine. This suggests that the current mechanism explaining
furosemide-induced urine acidification may not be correct, because other
methods for increasing Na+ delivery to the distal nephron do not acidify urine.
Thus, this study solidifies our previous finding that H+ secretion by furosemide
takes place already in the TAL and will contribute to a better understanding of
how the kidneys secrete acid.

The effect of P2-receptor inhibition on the cytotoxic effects of a-
haemolysin from E. coli – a murine sepsis model
Anne-Sofie Greve Christensen, Skals M, Praetorius HA
Aarhus University, Department of Biomedicine, Aarhus, Denmark
Haemolytic bacterial toxins cause cell lysis by forming pores in plasma
membranes. However, we demonstrated that a-haemolysin (HlyA) from E. coli
requires extracellular ATP and P2X receptor activation to cause lysis. Murine
erythrocytes express both P2X1 and P2X7 and inhibition of both receptor
subtypes completely prevent the HlyA-induced haemolysis. Interestingly, free
plasma haemoglobin is partly responsible for the symptoms during sepsis and is
associated with a poorer outcome of sepsis both in mice and in patients.
Inhibition of P2X receptors will improve the outcome of experimental induced
sepsis with HlyA producing E. coli in a murine model.
Iv-injection of HlyA producing E. coli or vehicle in anaesthetized mice for
determination of the role of P2X-receptors on the outcome of sepsis. Mice are
pre-treated with subcutaneous injection of P2X-receptor antagonists. Body
temperature and blood pressure are measured continuously. The mice are either
terminated after 2.5 hours, where blood is collected for determination of
haemolysis and plasma levels of IL1ß, TNFa, IL-6 and KC (murine equivalent of
IL-8) or allowed to die spontaneously of the bacteraemia (under anaesthesia).
Mice subjected to iv-injection of HlyA producing E. coli show an increased body
temperature and pulse pressure. Mice subjected to bacteria showed a massive
increase in the plasma levels of IL1ß ( 23 times increase), TNFa (459 times
increase), IL-6 (229 times increase) and IL-8 ( 48 times increase). Neither
haemolysis nor the cytokine release was statistically significantly different in
mice pre-treated with non-selective P2X receptor antagonist BBG (50 mg/kg,
resulting in a plasma level of 3 µM) compared to vehicle. Notably, 3 µM BBG is
sufficient to completely prevent HlyA-induced haemolysis in murine erythrocytes.
The survival was also comparable between BBG injected and control, with a
small trend towards longer survival of the BBG treated mice. Surprisingly, the
haemolysis is massively increased in P2X7 knockout mice compared to wild
type, p=0.0062. Moreover, our preliminary results show a markedly lower
survival of the P2X7 receptor knockout mice.
There is an apparent susceptibility of P2X7 receptor knockout mice for acute
bacteraemia. Since non-specific inhibition of P2X receptors show a different
profile from the P2X7 knock out mice, it is interesting to investigate specific
outcome in the absence of functional P2X1 receptors. These data may have
important clinical perspective, since loss of function mutations of the P2X7
receptor are relatively common in humans.

Luminal acid/base transporting proteins in the choroid plexus epithelium
Henriette Christensen, Damkier HH, Praetorius J
Aarhus University, Biomedicine, Denmark
Cerebrospinal fluid (CSF) pH most likely has a direct effect on brain extracellular
fluid pH, and disturbances in brain extracellular fluid pH have been shown to
affect neuronal excitability and cause symptoms such as seizures and panic
attacks. Little is known about the mechanisms behind CSF pH control. The CSF
is produced by the choroid plexus epithelium (CPE), located in the ventricular
system of the brain. It is believed that CSF pH is regulated by the CPE but the
mechanism is not yet known.
We detect V-ATPase-encoding mRNAs in fluorescence-activated cell sorted
(FACS) CPE samples. Immunofluorescence staining shows the protein to be
evenly dispersed across the cytoplasm in CPE cells and mass spectrometry on
surface-biotinylated CPE cells shows the presence of several V-ATPase
subunits in the luminal membrane. However, in vitro intracellular pH
measurements on freshly isolated CPE cells show no effect of the V-ATPase
inhibitor Concanamycin A on acid extrusion following an acid load. The alpha
chain of the non-gastric H+/K+-ATPase (HK α2) is also detected by mass
spectrometry on surface-biotinylated CPE cells, and immunohistochemistry
supports these data by locating the protein to the luminal CPE membrane.
However, HK α2-encoding mRNA cannot be detected in FACS CPE samples
and no beta chain has been identified at this site. We detect NHE6 and NHE1
mRNA by RT-PCR on FACS CPE samples and both proteins have been
detected by mass spectrometry on surface-biotinylated CPE cells, indicating
their presence in the luminal CPE membrane. Previous functional studies on
freshly isolated CPE cells from NHE1 knockout mice have shown that sodium-
dependent, and bicarbonate-independent acid extrusion was almost completely
abolished in these mice as compared to wild type mice. NBCe2-encoding mRNA
is readily detected in FACS CPE samples as well as by mass spectrometry on
surface-biotinylated CPE cells, and previous studies has shown the sodium-
bicarbonate cotransporter to be located in the luminal CPE membrane, where it
mediates the export of sodium and bicarbonate into the CSF. Thus, to date
luminal NHE activity is the most probable mechanism for CSF acidification by the
choroid plexus.

P2X receptor desensitization by high concentrations of ATP changes the
survival rate of THP-1 monocytes attacked by RTX toxins HlyA and LtxA
Steen K. Fagerberg, Skals M, Leipziger J, Praetorius HA
Aarhus University, Biomedicine, Denmark
The Repeats in Toxins (RTX) exotoxin alpha-haemolysin (HlyA) from
Escherichia coli is an important virulence factor for ascending urinary tract
infections. The RTX exotoxin LeukotoxinA from Actinobaccilus
actinomycetemcomitans is known to be involved in aggressive forms of
peridontitis, but is relevant for some systemic reactions as well.
The extracellular signalling molecule, adenosine triphosphate (ATP) has been
shown to play an important role in the erythrocyte damage inflicted by HlyA and
LtxA, and the subsequent recognition and phagocytosis of HlyA-attacked
erythrocytes by the monocytic cell line THP-1.
It is, however, uncertain how HlyA and LtXA affect the monocytes themselves.
Here we show that HlyA and LtxA initate two different patterns of [Ca2+]i
signalling combined of both immediate influx of Ca2+ trough the toxin pore and
activation of P2X and P2Y receptors by released ATP. Moreover we investigate
the THP-1 cell resistance to HlyA and LtxA and found that blockage of
P2Xreceptors by either desensitization with high concentrations of ATP or
blockage by oxATP resulted in increased survival of HlyA-attacked THP-1 cells
and a decreased survival of LtxA-attacked THP-1 cells.
In combination with the optimizing effect on erythrocytes of HlyA, these new
findings suggest an important interplay between monocytes, erythrocytes and
HlyA in situations of high amount of extracellular ATP, which may be relevant for
the development of cell lysis and cell recognition, at the place of injury.

The purinergic P2X7receptor is involved in glucose stimulated ATP release
and ß-cell proliferation
Anna Thorsø Larsen, Andersen MN, Novak I
Cell Biology and Physiology, Department of Biology, University of Copenhagen,
Denmark
Adenosine triphosphate (ATP) is not only important as an intracellular energy
source, but extracellularly it acts as a short-range signalling molecule that
promotes a broad range of physiological responses by activating purinergic
receptors on the cell membrane. Various subtypes of purinergic receptors are
found in pancreatic ß-cells and ATP act on these receptors regulating insulin
release, in some cases even in non-stimulating glucose concentration. In
addition, a few studies indicate that P2 purinergic receptors may also regulate ß-
cell survival, which is highly relevant to type 1 and 2 diabetes. One of the
receptors that may be relevant to cell survival is the P2X7 receptor, which is
expressed in ß-cells, but its detail role in ß-cell physiology is unclear. A recent
study indicates that high glucose can lead to extracellular ATP release in mouse
ß-cells. It is of utmost relevance to understand molecular mechanism regulating
ATP release and ß-cell survival.
The aim of this study was to determine whether extracellular glucose load
influences P2X7 receptor signalling in pancreatic ß-cell and determine how this
signalling can modulate ß-cell function. For this purpose we used pancreatic ß-
cell line INS-1e and in situ live cell luminometry to monitor ATP release and
BrdU assay to monitor proliferation. We show INS-1e releases ATP in response
increase in glucose (from 4-5mM to 16,7-25mM) and that this release is reduced
both by the P2X7 receptor inhibitor AZ10606120 and an inhibitor of the
transmembrane channel pannexin-1. Cell proliferation assays showed that
stimulation with ATP or the specific P2X7 agonist BzATP resulted in increased
cell proliferation. In addition, cell proliferation stimulated by high glucose was
markedly reduced by P2X7 receptor inhibition.
In conclusion, our study shows that the P2X7 receptor might be an important
regulator of ß-cell proliferation and ATP release.

Is megalin involved in regulating the apoptotic/anti-apoptotic apparatus of
melanoma cells?
Gitte T. Nielsen, Andersen, RK and Madsen M
Department of Biomedicine, Aarhus University, Denmark
The multiligand endocytic receptor megalin is a transmembrane protein known to
mediate cellular uptake and trafficking of various ligands. Amongst the many
established ligands for megalin is clusterin, suggested to play a role during
cellular protection from programmed cell death (apoptosis), which is particularly
relevant in cancer cells.
Interestingly, a recent study performed in our research group showed that
megalin is frequently expressed in melanoma tumors and metastases here off.
For comparison, megalin was only rarely observed in benign counterparts. In
addition, the same study demonstrated that siRNA-mediated knockdown of
megalin expression affects melanoma cell survival and induces apoptosis.
Megalin knockdown results in a significantly increased number of apoptotic cells
in megalin knockdown cultures compared to the number of cells in cultures
treated with non-targeting siRNA. This points towards a role for megalin in
regulating the apoptotic/anti-apoptotic apparatus of melanoma cells.
To further investigate the observed effect of megalin knockdown on melanoma
cell culture survival rates, this study aims to investigate a potential relation
between megalin expression and/or function and expression levels and
phosphorylation status of the protein Bcl2; which is known to operate during
cellular protection from apoptosis.
Preliminary results indicate that siRNA-mediated knockdown of megalin in
melanoma cell cultures causes a decrease in the amount of Bcl2-encoding
mRNA, which eventually can be speculated to lead to less Bcl2 protein and less
protection from apoptosis. The phosphorylation status of the remaining Bcl2
protein is currently being investigated and compared to the phosphorylation
status of Bcl2 protein in control cultures because phosphorylation of Bcl2 is
known to regulate its anti-apoptotic activity.

Cotransporter-mediated cerebrospinal fluid formation
Annette B. Steffensen, Damkier HH, Tritsaris K, Prætorius J and MacAulay N
Institute for Cellular and Molecular Medicine, University of Copenhagen,
Denmark
Cerebrospinal fluid (CSF) production takes place at around 500 ml/day in adult
humans. The majority of the CSF is produced in choroid plexus (CP) which is a
highly specialized cell layer with a vast variety of membrane transport proteins
expressed in its cell membrane, one of which is the K+/Cl- cotransporter (KCC). It
has been of long-standing scientific interest to resolve the mechanisms of CSF
production but the molecular players involved have remained elusive. As CSF
production readily takes place against an osmotic gradient and is only slightly
reduced by genetic deletion of AQP1, conventional osmotically driven water
transport does not appear to underlie CSF production. This project aims to
resolve the role of the KCCs and their ability to translocate water as the
molecular mechanism responsible for cerebrospinal fluid formation.
Localization and semi-quantification of the different KCC isoforms in mice
choroid plexus is addressed by immunohistochemistry, Western Blotting of intact
choroid plexus in addition to FACS-purified choroid plexus epithelial cells, and
quantitative PCR of FACS-purified choroid plexus epithelial cells. The extent to
which KCC activity participates in CSF production is assessed ex vivo by live cell
imaging of calcein-loaded mice choroid plexus, while the ability of the KCCs to
directly transport water will be quantified upon heterologous expression in the
Xenopus laevis expression system. Activation of KCC in the luminal membrane
of choroid plexus induced water transport against an imposed osmotic gradient
which indicates that KCC would indeed be able to drive water transport, i.e. CSF
production, independently of the osmolarity of the ventricular cerebrospinal fluid.
Although this study is ongoing, our preliminary data suggests that CSF is
produced by cotransport proteins, such as the K+/Cl- cotransporter, that directly
couple ion transport to water translocation.

Does the cytosolic adaptor protein Dab2 assist melanoma megalin during
endocytosis?
Mai-Britt H Thomsen, Rikke K Andersen and Mette Madsen, Department of
Biomedicine, Aarhus University, Denmark
Our research group recently showed that malignant melanoma tumors and
metastases hereof frequently express megalin, while benign counterparts only
rarely express megalin. Our study demonstrated that melanoma megalin
localizes partly to the plasma membrane and extensively to intracellular vesicular
structures. Interestingly, we were able to show that melanoma megalin can be
internalized from the cell surface to intracellular vesicles and it can colocalize
with a marker of early endosomes; Rab5, indicating a role for melanoma megalin
in endocytosis. A crucial role for melanoma megalin in uptake and/or trafficking
of ligands was further suggested from the data we obtained when megalin
expression was lowered by treatment with siRNA targeting the gene encoding
megalin. This had severe effects and decreased both proliferation and survival
rates dramatically. The cytosolic adaptor protein Disabled-2 (Dab2) is involved in
clathrin-mediated endocytosis of specific cargo proteins. Dab2 has the ability to
bind directly to clathrin and assist the assembly of the characteristic clathrin coat
surrounding endocytic vesicles. Simultaneously, it can recruit selected cargo to
these clathrin-coated vesicles thus; it functions as a clathrin-associated sorting
protein. One of the cargo proteins binding to Dab2 is megalin. To further
delineate the potential involvement of melanoma megalin in clathrin-mediated
endocytosis and vesicular trafficking of ligands, this project aims to investigate
the functional relationship between megalin and Dab2 in malignant melanoma
cells. A protocol for effective siRNA-mediated knockdown of Dab2 in melanoma
cells has already been established and our preliminary results indicate that
successful knockdown of Dab2 can be achieved both at RNA and protein level
within 48-72 hours. Most interestingly, Dab2 knockdown appears to have a
similar negative effect on melanoma cell culture viability as megalin knockdown
does indicating that these two proteins might be of similar importance to
melanoma cell maintenance. We are currently investigating in further detail the
effects of Dab2 knockdown on melanoma cell culture proliferation and survival
rates for comparison with the pattern observed from megalin knockdown studies.
Finally, the ultimate aim is to establish if Dab2 is essentially the sorting adaptor
protein required for melanoma megalin function.

Source: http://membranes.au.dk/fileadmin/membranes/Program_Sandbjerg_2015_final.pdf