Camerino, Giulia M.

Capogrosso, Roberta F.

Cozzoli, Anna

De Luca, Annamaria

Dinardo, Maria M.

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Disease-Related Modification of Chloride Conductance in Skeletal Muscle of Dystrophic Mice: Expression of CLC-1 Channel and Role of Inflammation and Oxidative Stress-Related Signaling 

Tuesday, February 28, 2012 549ashow that CLC-K channels are also inhibited at basic pH with almost complete
block at pH 11. Based on the structure of a bacterial homologue, we mutated all
external, basic, titratable residues of ClC-Ka (Arg, Lys, His, Cys, Tyr) and
identified K165, a highly conserved residue in the extracellular vestibule of
the channel, as the only candidate to be responsible for basic pH modulation.
Among the mutants K165A, K165C, K165H, K165Q, and K165R, only
K165R yielded (small) currents, with a weakened sensitivity to basic pH. We
obtained functional recovery of K165C by reaction with the positively charged
MTSEA reagent. Reversed voltage dependence of the alkaline pH effect on
MTSEA-modified K165C channels compared to WT, supported the involve-
ment of K165 in the pH modulation. The homologue K165C mutant of rat
ClC-K1 yielded currents which were less sensitive to basic pH than WT
CLC-K1 and MTSEA-modified CLC-K1-K165C channels recovered the WT
sensitivity to basic pH, confirming that block of CLC-K channels at alkaline
pH is mediated by the deprotonation of the pore lysine K165.
2791-Pos Board B561
Fluoride Transport in a Strange Subclass of Bacterial CLC Proteins
Randy Stockbridge, Christopher Miller.
HHMI, Brandeis University, Waltham, MA, USA.
Among prokaryotes, CLC-type Cl- channels and transporters are phylogeneti-
cally abundant but functionally obscure. Enteric bacteria such as E. coli are
known to use CLCs in extreme acid resistance, but many bacteria have several
CLC genes from far-flung branches of the phylogenetic tree. We have been
studying an evolutionarily distant CLC subclass that in sequence alignments
lacks the canonical, mechanistically crucial central serine, which coordinates
the central Cl- ion in E. coli CLC-ec1 and other well-studied homologues.
The protein-level function of these ‘‘strange’’ CLCs, whose genes have re-
cently been shown to be specifically upregulated by F- ion (J.L. Baker et al.,
in press), is unknown. Here we describe the overexpression, purification, and
functional reconstitution of several CLC homologues that appear to protect di-
verse bacterial species from F- toxicity. These ‘‘fluoride-CLCs’’ catalyze robust
F- transport in liposomes, as assayed by osmotic responses, F-/Cl- exchange,
planar bilayer recording, and 19F-NMR.We are currently carrying out a detailed
characterization of one of these homologues, CLC-ps from the plant pathogen
Pseudomonas syringae, including quaternary architecture, anion selectivity,
transport rate and mechanism, and the presence or absence of Hþ coupling.
2792-Pos Board B562
Age-Dependence of Chloride Currents in Muscle Fibers from Control and
Myotonic (HSALR) Mice
Carl H. Yu, Marino DiFranco, Marbella Quinonez, Julio L. Vergara.
UCLA, Los Angeles, CA, USA.
The transgenicmouseHSALR has been proposed as amodel formyotonic dystro-
phy type 1, capable of recapitulating the human pathology, including the loss of
chloride currents (ICl). Since the structural organization of the transverse tubular
system (TTS) is not completed until ~3 weeks after birth, and the functional ex-
pressionofClC-1 channelsmaydepend on this,wewanted to investigatewhether
the reduction of ICl in HSA
LR animals, compared to that in normal controls,
changeswith age. To this end,we performed a longitudinal study of the evolution
of ICl and TTS voltage transients (using the potentiometric dye di-8-ANEPPS) in
fibers from 2, 3, 4, 16 and 26 weeks old animals. ICl and optical signals were si-
multaneously acquired in fibers under voltage-clamp conditions using a 3-pulse
protocol and a 2-microelectrode system. Thefiberswere bathed in 156mMTEA-
Cl and internally equilibratedwith 70mMCl. The characteristics of ICl, and their
effects on di-8-ANEPPS transients, were predicted using a radial cable model of
the TTS. Our results show that, whereas fibers from 2-week oldHSALRmice dis-
played peak ICl (for a120mV pulse) of110550 mA/cm2 (n=8), their control
counterparts’ were510580 mA/cm2 (n=6). Furthermore, the magnitude of ICl
increases with age in both animal strains, and by 16 weeks they reach similar
levels (>750 mA/cm2). Thus, a reduction in ICl is significant in very young
HSALR specimens, but not apparent in mature animals. We also verified that in
HSALR animals the gain in total ICl with age is accompanied by the expression
of functional ClC-1 channels in the TTS system. This work was supported by
NIH grants AR047664, AR041802, and AR054816. HSALR mice were kindly
provided by Dr. C. Thornton, University of Rochester.
2793-Pos Board B563
Functional Regulation of ClC-3 in the Migration of Vascular Smooth
Muscle Cells
Sindura B. Ganapathi1, Shun-Guang Wei2, Fred S. Lamb3,
Stephen B. Shears1.
1National Institutes of Environmental Health Sciences, Research Triangle
Park, NC, USA, 2Department of Internal Medicine, University of Iowa
Carver College of Medicine, Iowa City, IA, USA, 3Vanderbilt University,
Nashville, TN, USA.Migration of vascular smooth muscle cells (VSMC) into neointima contributes
to hypertension, atherosclerosis, and restenosis. Cell migration requires co-
ordinated plasmalemmal fluxes of water and ions. Here, we show that regula-
tion of transmembrane Cl- flux significantly impacts the migration of VSMC.
Gene disruption of ClC-3, a Cl- channel/transporter, halved the rate of cell mi-
gration in transwell assays. Functional regulation of plasmalemmal Cl- current
by ClC-3 was studied by electrophysiological recordings. Raising intracellular
[Ca2þ] from zero to 0.5 mM in wild-type cells stimulated a Cl- current (ICl.Ca)
that was reduced approximately 40% upon CaMKII inhibition by 100 mMKN-
93, or by application of 10 mM inositol-3,4,5,6-tetrakisphosphate, a cellular sig-
nal that specifically prevents CaMKII from activating ICl.Ca. Thus, ICl.Ca com-
prises two components, one directly activated by Ca2þ, and another that
requires CaMKII. ICl.Ca was 50% smaller in ClC-3 null cells compared to
wild-type VSMC; neither KN-93 nor inositol-3,4,5,6-tetrakisphosphate af-
fected ICl.Ca in ClC-3 null VSMC. Thus, the CaMKII-mediated component of
ICl.Ca in VSMC is dependent upon ClC-3. Both Ca
2þ-dependent and
CaMKII-dependent forms of ICl.Ca were strongly inhibited by niflumic acid,
a Cl- channel blocker, but, significantly, that drug only inhibited migration of
wild-type and not ClC-3 null VSMC. Moreover, a cell-permeant, bio-activat-
able analogue of inositol-3,4,5,6-tetrakisphosphate inhibited migration in
wild-type, but not ClC-3 null cells. Our work describes for the first time a spe-
cific role of ClC-3 in VSMC migration, thereby revealing new therapeutic di-
rections in vascular remodeling diseases.
2794-Pos Board B564
Disease-Related Modification of Chloride Conductance in Skeletal Muscle
of Dystrophic Mice: Expression of CLC-1 Channel and Role of Inflamma-
tion and Oxidative Stress-Related Signaling
Giulia M. Camerino, Anna Cozzoli, Roberta F. Capogrosso,
Maria M. Dinardo, Annamaria De Luca.
Unit of Pharmacology, Dept. Pharmacobiology, University of Bari, Italy.
A decrease in resting chloride channel conductance (gCl) occurs in dystrophin-
deficient myofibers of mdx mouse as a consequence of both spontaneous de-
generation, as in diaphragm (DIA), or exercise-induced damage as in fast-
twitch EDL muscle (De Luca et al., J. Pharmacol. Exp. Ther. 2003). Alterations
in gCl, in parallel with markers of inflammation, can be contrasted by small res-
toration in dystrophin expression (De Luca et al., Neurobiol Dis, 2008). We fo-
cused on the molecular mechanisms underlying gCl impairment and its relation
to primary defect. Preliminary qRT-PCR experiments showed a 30-35% reduc-
tion of CLC-1 mRNA in both DIA and EDL muscles of mdx mice, irrespective
to exercise regimen. The reduction was consistent with the impairment of gCl
detected in concomitant electrophysiological experiments in DIA and EDLmy-
ofibers; however the selective alteration of gCl in exercised mdx EDL muscle
remained unexplained. Further experiments are ongoing to evaluate the possi-
ble outcome of pathology and mechanical stress on CLC-1 channel protein
level and on the expression of other chloride channel types. Recent results
showed that in vivo inhibition of angiotensin (Ang)-II contrasts the decrease
in gCl in mdx EDL muscle, disclosing a possible role of this pro-
inflammatory and pro-oxidative mediator in chloride channel function (Cozzoli
et al., Pharmacol Res 2011). The application of Ang-II to wt EDL fibers re-
duced gCl in a concentration-dependent manner, with a half-maximal concen-
tration of 67nM. The effect was inhibited by the AT1-receptor antagonist
losartan, as well as by the PKC-inhibitor chelerythrine, the antioxidant N-ace-
tyl-cysteine and the inhibitor of NADPH-oxidase apocynin. The results demon-
strate that CLC-1 channel expression is affected in dystrophinopathies and that
further modulation of gCl is related to inflammation and oxidative stress in
skeletal muscle.
2795-Pos Board B565
Single Molecule Optical Determination of Bestrophin Stoichiometry
Shashank Bharill, Ehud Y. Isacoff.
University of California Berkeley, Berkeley, CA, USA.
Best macular dystrophy (BMD) is an autosomal dominant form of macular de-
generation, linked to mutations in the BEST1 gene that encodes the calcium ac-
tivated chloride channel, bestrophin-1. BEST1 belongs to the bestrophin family
of anion channels. It is a 585 amino acid transmembrane protein localized to the
basolateral membrane of the retinal pigment epithelium (RPE). Structurally,
BEST1 has been suggested to have 4 transmembrane segments, with intracel-
lular N and C termini. The stoichiometry of BEST1 is not clear, having been
suggested to be dimers in hydrodynamic studies of porcine BEST1 in
TritonX-100 and tetramers or pentamers in coimmunoprecipitation studies of
human BEST1 (hBEST1). In this study, we employed our single molecule
method to determine subunit number by counting the number of fluorescence
bleaching steps of eGFP tagged hBEST1. The advantage of this method is
that the counting can be done on the cell surface of live cells (Xenopus oocytes


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Disease-Related Modification of Chloride Conductance in Skeletal Muscle of Dystrophic Mice: Expression of CLC-1 Channel and Role of Inflammation and Oxidative Stress-Related Signaling

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