Brett, Tom J.

Nichols, Colin G.

Sala-Rabanal, Monica

Yurtsever, Zeynep

Elsevier - Publisher Connector 

Wednesday, February 19, 2014 751aadvantage of this we used electrophysiological techniques on HEK293 cells ex-
pressing Piezo channels to investigate the effects of Phospholipase C (PLC)
activating pathways and specifically phosphoinositide depletion. PLC activa-
tion, which converts PIP2 into IP3 and DAG, inhibited mechanically activated
currents in Piezo expressing cells. Phosphoinositide depletion using rapidly
inducible lipid phosphatases also inhibited Piezo channel activity, but without
generating IP3 or DAG. Our data provides evidence that phosphoinositides are
involved in the regulation of Piezo channels, thus our study contributes to the
understanding of lipid regulation of mechanically gated channels.
3797-Pos Board B525
Linolenic and Linoleic Acid Induce the Opening of Connexin 43, 46 and 50
Hemichannel in Human Hela Cells
Vania A. Figueroa, Mauricio A. Retamal.
Physiology, Universidad del Desarrollo, Santiago, Chile.
Abstract: Connexin (Cx) hemichannels (HC) located at the plasma membrane
have a very low open probability, but under physiological conditions they can
briefly open, allowing passage of both ions and small signaling molecules,
thereby participating in the control of diverse cellular responses. Cx43, 46 and
50 are expressed in human lens and are essential to maintaining its transparency,
since mutations in the genes coding for these Cxs have been identified, inducing
the development of different types of cataracts. However, non-congenital cata-
racts may result from consumption of polyunsaturated fatty acids (PUFAs).
While the consumption of omega-6 PUFAs, such as linoleic acid (u-6LA) has
been associated with development of cataracts, intake of omega-3linolenic
acid (u-3LA) has been associated with their decline. Here, we report the effects
of u-6LA and u-3LA on HCs in HeLa cells that express Cx43, Cx46 or Cx50.
Bath application of u-6LA or u-3LA induces opening of HCs (measured as
Ethidium uptake) formed by Cx43, Cx46 or Cx50in HeLa cells and also in hu-
man lens epithelial cell line (HLE) that expresses endogenously Cx46 and
Cx50 HCs, in a concentration- dependent manner and inhibited by HC blockers
such as Lanthanum. Both PUFAs also increased themembrane current mediated
by these HCs measured by patch clamp assays in the whole cell configuration in
both cell types. Since PUFAs have demonstrated cytotoxic effects in HLE and
also in organotypic cultures of bovine lens epithelial cells, their effect on HCs
might be relevant to development and prevention of cataracts.
3798-Pos Board B526
Enhancing the ATPase Activity of the Cystic Fibrosis Transmembrane
Conductance Regulator CFTR in Live Cells Reduces Chloride Flux
Anna Seelig, Matthias Zwick.
Biozentrum, University of Basel, Basel, Switzerland.
Maintaining the fluid balance in tissues such as the lung airways, the intestine,
and many exocrine glands is of vital importance. This task is fulfilled essen-
tially by the cystic fibrosis transmembrane conductance regulator (CFTR,
ABCC7), a channel for passive diffusion of chloride and bicarbonate. Deregu-
lation of CFTR causes multiple disorders, the most prominent of which is cystic
fibrosis (CF). CFTR is also implicated in autosomal dominant polycystic kid-
ney disease and secretory diarrheas. Owing to the increasing number of dis-
eases recognized to be associated with CFTR dysfunction, the interest in
CFTR potentiators and inhibitors has grown. However, their function at the mo-
lecular level is not yet understood. On the one hand, this is due to the complex
function of CFTR which, as a member of the ATP binding cassette (ABC)
transporter family, is the only known ion channel that links ATP binding and
hydrolysis to channel gating; on the other hand, it is due to fact that chloride
efflux is generally measured in cells, whereas ATP hydrolysis was so far
measured in reconstituted proteoliposomes exhibiting significant functional
attrition. Here we take a new approach to answer the intriguing question as
to whether chloride channel inhibitors and potentiators enhance or reduce
ATP hydrolysis by CFTR. For this purpose we measured ATPase activity
and chloride efflux, both, in live cells, under similar experimental conditions.
ATPase activity was measured by microphsiometry using silicon chip technol-
ogy and anion flux was measured using an iodide electrode. Most importantly
we demonstrate that chloride flux inhibitors enhance ATPase activity of CFTR,
whereas chloride channel potentiators do not stimulate ATPase activity, but
tend to reduce cell metabolism. These findings have implications for the syn-
thesis of new potentiators and inhibitors.
3799-Pos Board B527
MechanismofCLCA1-MediatedCACCActivation inChronicLungDiseases
Zeynep Yurtsever, Monica Sala-Rabanal, Colin G. Nichols, Tom J. Brett.
Biochemistry, Washington University in St Louis, St. Louis, MO, USA.
CLCA1 plays a central role in chronic inflammatory lung diseases (asthma and
COPD). Overexpression of CLCA1 drives mucous overproduction and acti-
vates a Ca-activated chloride channel (CaCC), both through unknown mecha-
nisms. Here we show that CLCA1 is a soluble secreted protein that gets cleavedinto two fragments upon secretion. The cleavage is carried out by a novel inter-
nal zincin metalloprotease, located in the N-terminal domain. This self-
cleavage reaction is required for the activation of chloride currents, as mutants
with an impaired active site or cleavage site failed to increase currents above
background levels. We observed that the N-terminal cleavage fragment is
necessary and sufficient to activate the unknown CaCC, without directly pro-
teolytically cleaving the channel. These results suggest a direct interaction be-
tween the channel and its modulator, CLCA1. Our current focus is on showing
physical and functional association between these two molecules and identi-
fying the CaCC by using pulldown experiments and functional patch clamp as-
says in the presence of siRNA and channel blockers. Latest results will be
presented.
3800-Pos Board B528
The Maguk Scaffolding Protein CASK Regulates TMEM16A Channel
Function by Phosphorylation
Kuai Yu, Jinqiu Zhu, Yuanyuan Cui, H. Criss Hartzell.
Emory University, Atlanta, GA, USA.
Ca-activated Cl channels (CaCCs) encoded by TMEM16A (Ano1) play essen-
tial roles in many physiological processes including epithelial fluid secretion,
gut motility, and smooth muscle tone. Using a quantitative SILAC proteomics
approach, we discovered that CASK, a unique MAGUK protein containing an
N-terminal CaM-kinase homology domain, is very highly enriched in the
TMEM16A interactome. CASK contains multiple protein-protein interaction
domains linking receptors and signaling molecules at membrane-cytoskeletal
interfaces and has unique protein kinase activity that is inhibited by Mg2þ.
Here we show that CASK co-immunoprecipitates with TMEM16A and regu-
lates the voltage-dependence of TMEM16A in Mg2þ-sensitive manner. In the
presence of 1 mM free intracellular Ca2þ, decreasing intracellular Mg2þ from
2 mM to 0 mM shifts the conductance-voltage (G-V) relationship 98.4 mV in a
hyperpolarizing direction without changing GMAX or Ca2þ sensitivity. The
Mg2þ-dependent shift in the G-V curve is dependent on CASK protein kinase
activity because shRNA-knockdown of CASK or replacement of ATP with
AMP-PNP eliminates the shift. Mutation of S639 in TMEM16A identifies
this amino acid is the site phosphorylated by CASK. The G-V curve of the
S639A mutant is unaffected by Mg2þ or ATP and has a V0.5 value similar
to WT in the presence of 2 mM Mg2þ [WT(þMg2þ) 98.6 mV;
S639A(0Mg2þ) 78.8 mV]. The G-V curve of the phosphomimetic S639E
mutant is unaffected by Mg2þ or ATP, but the V0.5 value is similar to WT
in the absence of Mg2þ [WT(0 Mg2þ) 3.5 mV; S639A(2 Mg2þ) 7.7 mV].
Inhibitory autocamtide-2 (AIP-2), a CaMKII inhibitory substrate that also in-
terferes with the catalytic activity of CASK in the internal solution efficiently
abolishes the effect of Mg2þ on TMEM16A currents. These data suggest that
CASK may regulate TMEM16A channels, although the physiological signif-
icance in cells remains to be determined.
3801-Pos Board B529
Modulation of the Voltage-Gated Proton Channel Hv1 by Small Gating-
Modifier Ligands
Bosmat Refaeli, Polina Kornilov, Asher Peretz, Bernard Attali.
Physiology, Sackler Medical School, Tel Aviv University, TelAviv, Israel.
The human voltage-gated proton channel Hv1 is activated by depolarization
and intracellular acidification to maintain the cell’s neutral pH. It belongs to
voltage-gated cation channels, consisting of a typical voltage sensor domain
(VSD) but lacking a canonical pore domain. These channels assemble as di-
mers and recent data indicate strong cooperativity between the two subunits.
Recently, guanidine derivatives were found to act as open channel blockers
on the Hv1 VSD. Here we show that the gating modifiers NH29 and NH34,
two diphenylamine carboxylate derivatives, act as Hv1 channel openers.
NH29 was recently shown to exert opposite gating-modifier effects on the
VSD of Kv7.2 potassium channels and TRPV1 cationic channels operating
as opener and blocker of Kv7.2 and TRPV1 currents, respectively. Both
compounds increase the Hv1 current amplitude by more than 2.5-fold at þ
50 mV and produce a left-shift in the voltage dependence of channel activa-
tion by about 15 mV. We found that NH29 and NH34 change the proton
selectivity of Hv1 channels and produce a left-shift of about 10 mV in
the reversal potentials both in symmetrical (pH 7.0) and asymmetrical pH so-
lutions (pHi 5.5/pHo 7.0). Our results also show that the mutation R211S in
S4 profoundly affects the modulatory properties of the compounds. The S4
residue R211 was previously suggested to be crucial for Hv1 proton selec-
tivity by interacting with D112 in S1. In mutant R211S, NH29 does not
act anymore as an opener but rather inhibits Hv1 currents. Other Hv1 muta-
tions are currently tested in order to determine how Hv1 channels interact
with these small ligands and to elucidate Hv1 selectivity and gating mecha-
nisms.


Mechanism of CLCA1-Mediated CACC Activation in Chronic Lung Diseases 

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Mechanism of CLCA1-Mediated CACC Activation in Chronic Lung Diseases

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