0022-3565/07/3213-1075–1084$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics
JPET 321:1075–1084, 2007
Vol. 321, No. 3
118786/3206608
Printed in U.S.A.
Stimulation of Ca2⫹-Gated Cl⫺ Currents by the CalciumDependent K⫹ Channel Modulators NS1619 [1,3-Dihydro-1-[2hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2Hbenzimidazol-2-one] and Isopimaric Acid
Sohag N. Saleh, Jeff E. Angermann, William R. Sones, Normand Leblanc, and
Iain A. Greenwood
Received December 19, 2006; accepted March 5, 2007
ABSTRACT
Because chloride (Cl⫺) channel blockers such as niflumic acid
enhance large-conductance Ca2⫹-activated potassium channels (BKCa), the aim of this study was to determine whether
there is a reciprocal modification of Ca2⫹-activated chloride
Cl⫺ currents (IClCa) by two selective activators of BKCa. Single
smooth muscle cells were isolated by enzymatic digestion from
murine portal vein and rabbit pulmonary artery. The BKCa activators NS1619 [1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one] and isopimaric acid (IpA) augmented macroscopic IClCa elicited by
pipette solutions containing [Ca2⫹]i ⬎ 100 nM without any
alteration in current kinetics. Enhanced currents recorded in the
presence of NS1619 or IpA reversed at the theoretical Cl⫺
equilibrium potential, which was shifted by approximately ⫺40
mV upon replacement of the external anion with the more
permeable thiocyanate anion. NS1619 increased the sensitivity
of calcium-activated chloride channel (ClCa) to Ca2⫹ (⬃100 nM
at ⫹60 mV) and induced a leftward shift in their voltage dependence (⬃80 mV with 1 ␮M Ca2⫹). Single-channel experiments
revealed that NS1619 increased the number of open channels
times the open probability of small-conductance (1.8 –3.1 pS)
ClCa without any alteration in their unitary amplitude or number
of observable unitary levels of activity. These data, in addition
to the established stimulatory effects of niflumic acid on BKCa,
show that there is similarity in the pharmacology of calciumactivated chloride and potassium channels. Although nonspecific interactions are possible, one alternative hypothesis is that
the channel underlying vascular IClCa shares some structural
similarity to the BKCa or that the latter K⫹ channel physically
interacts with ClCa.
S.N.S. was funded by a Glaxo Smith Kline-Biotechnology and Biological
Sciences Research Council award. This work was supported by the National
Institutes of Health (Grant HL 1 R01 HL075477-01 to N.L.), by the British
Heart Foundation (Grant PG/05/038 to I.A.G.), and by The Wellcome Trust
(grant to I.A.G.). This publication was also made possible by Grant NCRR 5P20
RR15581 (to N.L.) from the National Center for Research Resources, a component of the National Institutes of Health supporting a Center of Biomedical
Research Excellence at the University of Nevada School of Medicine (Reno,
NV). Its contents are solely the responsibility of the authors and do not
necessarily represent the official views of National Center for Research Resources or National Institutes of Health.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.118786.
Ca2⫹-activated chloride Cl⫺ currents (IClCa) are present in
a wide variety of cell types, including endothelial, secretory,
and smooth muscle cells (Hartzell et al., 2005; Leblanc et al.,
2005). In vascular smooth muscle cells, IClCa have a number
of distinctive characteristics, including an activation threshold for [Ca2⫹] of about 200 nM, voltage-dependent kinetics, a
lyotropic anion permeability profile, and small unitary conductance (Hartzell et al., 2005; Leblanc et al., 2005). In
contrast to the extensive electrophysiological studies on
IClCa, the molecular identity of the gene(s) encoding for the
channel generating IClCa is still unknown, and attempts to
ABBREVIATIONS: IClCa, calcium-activated chloride current; NFA, niflumic acid; CCB, chloride channel blocker; BKCa, calcium-activated potassium channel; NS1619, 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one; IpA, isopimaric acid; PV,
portal vein; I-V, current-voltage; Iinitial, current measured at the beginning of a voltage-clamp test pulse immediately following the capacitative
current; Ilate, current measured at the end of a voltage-clamp test pulse; GClCa, conductance of the calcium-activated chloride channel; Erev,
reversal potential of the current; NP0, number of open channels times the open probability; Itail, current measured 20 ms after repolarization from
a voltage-clamp step used to elicit time-dependent calcium-activated chloride current; IBKCa, calcium-activated potassium current; ClCa,
calcium-activated chloride channel.
1075
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Ion Channels and Cell Signaling Research Centre, Division of Basic Medical Sciences, St. George’s, University of London,
London, United Kingdom (S.N.S., W.R.S., I.G.); and Department of Pharmacology, Center of Biomedical Research Excellence,
University of Nevada School of Medicine, Reno, Nevada (J.E.A., N.L.)
1076
Saleh et al.
tural similarity with the BKCa or that the latter channel
physically interacts with Ca2⫹-activated Cl⫺ channels.
Materials and Methods
Cell Dissociation and Solutions. IClCa were recorded using the
whole-cell voltage-clamp technique from single smooth muscle cells
isolated from murine PV or rabbit pulmonary artery. BALB/c mice
(6 – 8 weeks) were sacrificed by cervical dislocation in accordance
with schedule 1 of the United Kingdom Animals Act (1986). After an
incision of the abdomen, the PV was removed and immediately
placed in chilled physiological salt solution composed of 125 mM
NaCl, 5.4 mM KCl, 15.4 mM NaHCO3, 0.33 mM Na2HPO4, 0.34 mM
KH2PO4, 10 mM glucose, 11 mM HEPES, and 1 mM CaCl2 (pH was
adjusted to 7.2 with NaOH). The PV was freed of fat and connective
tissue and then cut into longitudinal strips and individual smooth
muscle myocytes isolated using the same procedure described by
Saleh and Greenwood (2005). Rabbit pulmonary artery myocytes
were prepared as described fully by Greenwood et al. (2001, 2004). A
small aliquot of smooth muscle cells stored in 0.1 to 0.01 mM CaCl2
physiological salt solution was placed in a glass chamber on the stage
of a Zeiss Axiovert (Carl Zeiss MicroImaging GmbH, Jena, Germany)
or Nikon Diaphot TMD inverted microscope (Nikon, Tokyo, Japan).
After allowing 15 to 20 min for adhesion, the cells were superfused at
a rate of 5 ml/min, with an external solution composed of 126 mM
NaCl, 11 mM glucose, 10 mM HEPES, 10 mM TEA-Cl, 1.2 mM
MgCl2, and 1.5 mM CaCl2 (pH was adjusted to 7.2 with NaOH).
Individual SMCs were identified by their spindle-shaped appearance
and ability to contract.
Electrophysiology and Statistical Analysis. Macroscopic IClCa
was recorded using the whole-cell configuration of the patch-clamp
technique with either an EPC-8 HEKA (Lambrecht/Pfalz, Germany)
or an Axopatch-1D (Molecular Devices, Sunnyvale, CA) patch-clamp
amplifier. Voltage-clamp protocols were computer-driven using a
D/A and A/D acquisition system (DigidataA 1322 board; Molecular
Devices) and pClamp 8.2 or 9.0 software (Molecular Devices). Patch
pipettes were manufactured from borosilicate glass and fire polished,
giving pipettes with resistance of between 2 and 5 M⍀. IClCa was
evoked using pipette solutions containing a known concentration of
free [Ca2⫹]. With this technique, pipette solutions containing free
[Ca2⫹] higher than the activation threshold (⬃180 nM) persistently
stimulate the Cl⫺ channel resulting in a sustained Cl⫺ current
without any reliance upon Ca2⫹ influx or release from internal stores
as described in a number of previous publications (Greenwood et al.,
2001, 2004; Britton et al., 2002; Piper et al., 2002; Angermann et al.,
2006). The intracellular solution contained 106 mM CsCl, 20 mM
TEA, 3 mM Na2ATP, 0.2 mM GTP䡠Na, 10 mM HEPES-CsOH, pH
7.2, 10 mM BAPTA, and 0.42 mM MgCl2 (0.42) and an appropriate
concentration of CaCl2 (0.84 – 8.4 mM) to make up free Ca2⫹ concentrations of 0.02, 0.1, 0.25, 0.5, and 1 ␮M as calculated by EqCal
(Biosoft, Ferguson, MO). Under these conditions, any contribution
from K⫹ channels should be negligible, and the membrane conductance is dominated by Cl⫺ channel activity. A number of different
voltage protocols were employed in the present study. A single test
step every 15 s from ⫺60 to ⫹90 mV for 750 ms followed by repolarization to ⫺80 mV for 500 ms was used to ascertain when IClCa had
stabilized and to investigate the time course of different drug effects
(see Fig. 2B). Once IClCa had stabilized, a control current-voltage
(I-V) relationship was constructed by depolarizing the myocyte from
⫺60 mV to a range of voltages from ⫺100 to ⫹100 mV for 1.5 s. I-V
relationships were constructed at two different time points, namely
immediately after the onset of the test step (Iinitial) and immediately
before termination of the test pulse (Ilate; for example, see Fig. 1B). In
Figs. 4 and 5, the chord conductance of IClCa in nanosiemens (GClCa)
was calculated using the following formulation (eq. 1):
G ClCa共V兲 ⫽ Ilate/共V ⫺ Erev)
(1)
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
identify the proteins underlying IClCa have been hampered by
the lack of high-affinity pharmacological probes. A number of
structurally disparate agents block IClCa in smooth muscle
cells (Large and Wang, 1996), with the most potent being
niflumic acid (NFA), which inhibits spontaneously occurring
transient Cl⫺ currents with an IC50 of approximately 2 ␮M
(Large and Wang, 1996). However, the inhibitory efficacy of
NFA and other putative Cl⫺ channel blockers (CCBs) such as
anthracene-9-carboxylic acid is inversely proportional to the
degree of Cl⫺ channel stimulation. Consequently, the IC50 for
NFA block of longer lasting IClCa evoked as a consequence of
promoting Ca2⫹ influx through voltage-dependent Ca2⫹
channels is about 15 ␮M (Leblanc et al., 2005; Saleh and
Greenwood, 2005). As a corollary to these findings, fully
activated or sustained IClCa evoked by pipette solutions of
known free [Ca2⫹] are not inhibited markedly by NFA at
concentrations [Ledoux et al., 2005; IC50 ⫽ 159 ␮M at ⫹60
mV with 500 nM intracellular Ca2⫹ concentration ([Ca2⫹]i)]
far higher than required to abolish spontaneously occurring
IClCa. Paradoxically, niflumic acid and similar agents actually stimulate sustained IClCa, which is more obvious upon
washout of the agent where IClCa increases by about 200 to
300% in rabbit pulmonary or coronary artery myocytes (Piper
et al., 2002; Piper and Greenwood, 2003; Ledoux et al., 2005).
These observations led to the suggestion that the Cl⫺ channel protein contained at least two binding sites for NFA: a
low-affinity inhibitory site and a high-affinity stimulatory
site that becomes more prominent with greater stimulation
of the channel (Ledoux et al., 2005).
In addition to its effect on IClCa NFA also stimulates largeconductance, Ca2⫹-dependent K⫹ channels (BKCa) in lipid
bilayers (e.g., Ottolia and Toro, 1994) and vascular smooth
muscle cells (Greenwood and Large, 1995). A number of
experiments in smooth muscle cells led to the postulate that
NFA and structurally similar agents, such as flufenamic acid
and mefenamic acid, acted as partial agonists at a calcium
binding domain on the BKCa complex (Greenwood and Large,
1995). Subsequent to this work, Toma et al. (1996) showed
that three other chemically distinct CCBs, anthracene-9-carboxylic acid, ethacrynic acid, and indanyloxyacetic acid, also
activated BKCa in vascular smooth muscle cells at concentrations higher than required to block IClCa. Consequently, there
seems to be similarity in the pharmacology of BKCa and the
channel underlying IClCa, especially in vascular myocytes.
The aim of the present study was to explore this possibility
further by studying how the well characterized BKCa modulators NS1619 and isopimaric acid (IpA) affect native IClCa in
vascular smooth muscle cells (for chemical structures, see
Olesen et al., 1994; Imaizumi et al., 2002). Because the structure of the BKCa is well characterized, the development of
these pharmacological investigations may give some clues
about structural features of the Cl⫺ channel to be deciphered.
Specifically, we investigated whether NS1619 and IpA,
which stimulate BKCa by a direct interaction with the poreforming subunit encoded by Slo1 (Joiner et al., 1998;
Imaizumi et al., 2002), affect IClCa elicited by fixed elevated
levels of [Ca2⫹]i in murine portal vein (PV) and rabbit pulmonary artery smooth muscle cells. Our data revealed that
the BKCa activators augmented IClCa, and this effect was
reversed by subsequent application NFA. These results suggest that the channel underlying IClCa may bear some struc-
Modulation of Calcium-Activated Chloride Channels
1077
where Ilate is the current measured at the end of the pulse (see Fig.
1B), V is the potential in mV that evoked Ilate, and Erev is the reversal
potential of Ilate determined by analysis of the I-V relationship.
Finally, a two-step protocol was employed to determine the reversal
potential of the currents evoked in the absence and presence of
different agents. Cells were initially depolarized from ⫺60 to ⫹90
mV to fully activate the channels and then stepped to different
test potentials between ⫺100 and ⫹40 mV at 20-s intervals (see
Fig. 2B).
For single-channel recordings of IClCa in the inside-out configuration, the bath solution contained 108 mM CsOH, 108 mM aspartic
acid, 18 mM CsCl, 10 mM glucose, 11 mM HEPES, 1.2 mM MgCl2, 1
mM Na2ATP, 0.2 mM Na2GTP, and CaCl2 (0.1 mM for 14 nM free
Ca2⫹ and 1.5 mM for 500 nM free Ca2⫹, calculated using EqCal
software; pH was adjusted to 7.2 using Trizma base). The pipette
solution facing the external surface of the membrane contained 126
mM N-methyl-D-glucamine Cl⫺ (prepared by equimolar addition of
N-methyl-D-glucamine and HCl), 1 mM glucose, 10 mM HEPES,
0.005 mM nicardipine, 10 mM TEA-Cl, 10 mM 4-aminopyridine,
0.0001 mM iberiotoxin, 1.2 mM MgCl2, and 5 mM CaCl2 (pH adjusted to 7.2 with N-methyl-D-glucamine or HCl as appropriate).
Unitary currents produced by the opening of single Ca2⫹-activated
Cl⫺ channels were recorded with a EPC-8 HEKA patch-clamp amplifier at room temperature using the inside-out patch configuration
of the patch-clamp technique. For these experiments, the micropipettes were fire-polished more heavily than those used for whole-cell
recording experiments, yielding a pipette resistance lying between
10 and 15 M⍀. To reduce line noise, the recording chamber was
superfused with solutions delivered through two 20-ml syringes, one
filled with external solution and the other used to drain the chamber,
in a “push and pull” technique. The external solution could thus be
exchanged twice within 30 s. When recording single-channel currents, the holding potential was set at ⫹80 mV, and to evaluate I-V
characteristics of unitary channel currents, the membrane potential
was manually changed between ⫺80 and ⫹80 mV by turning the
holding potential knob on the patch-clamp amplifier. Single-channel
currents were initially recorded onto digital audio tape using a
Fig. 2. NS1619 enhances a current whose properties are consistent with
those of IClCa in murine portal vein myocytes. A, three families of current
traces recorded in control, after a 3-min exposure to 30 ␮M NS1619, and 5
min following washout of the drug. Currents were elicited by 750-ms voltageclamp steps ranging from ⫺100 to ⫹120 mV (20-mV increments) from a
holding potential of ⫺50 mV. Bi, IClCa evoked by the double-pulse voltageclamp protocol shown below the two families of IClCa traces recorded in the
absence (filled square) and presence (open circle) of 30 ␮M NS1619. Bii,
graph, mean data from five similar experiments to Bi. The plots were
generated by measuring the amplitude of Itail 20 ms after stepping to the test
potential and plotted against the corresponding voltage step. The current
enhanced by NS1619 (open circles) reversed at the same voltage (⬃0 mV) as
IClCa recorded in the absence of drug (filled squares). Scalars, 200 pA and 500
ms. A and B, cells were dialyzed with 250 nM free [Ca2⫹].
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Fig. 1. Characteristics of sustained
IClCa evoked in murine portal vein
myocytes. A, families of currents recorded at potentials between ⫺100
and ⫹120 mV following activation of
IClCa by pipette solutions of different
[Ca2⫹] (as shown). B, example of the
effect of a test step to ⫹90 mV on the
sustained IClCa evoked by 500 nM free
[Ca2⫹]. B highlights the different parameters measured during the study.
Protocol shown as an insert. Immediately after membrane depolarization,
there was an instantaneous outward
current (Iinitial) that was followed by a
slowly developing outward relaxation
resulting in more outward current at
the end of the test step (Ilate). Upon
repolarization to ⫺80 mV, an exponentially declining inward current
(Itail) was recorded. Dashed line, zero
current level. C, mean late current
amplitude at five different test potentials plotted against activator [Ca2⫹]
and fitted by a standard logistic equation. Data are the mean of ⬎four cells.
1078
Saleh et al.
Bio-Logic DRA-200 digital tape recorder (Bio-Logic SAS, Claix,
France) at a bandwidth of 5 kHz (⫺3 db, low-pass four-pole Bessel
filter, HEKA EPC-8 patch-clamp amplifier) and a sampling rate of 48
kHz. For off-line analysis, single-channel records were filtered at 100
Hz (⫺3 db, low-pass eight-pole Bessel filter, Frequency Devices,
model LP02; Scensys Ltd,. Aylesbury, UK) and acquired using a
Digidata 1322A and pCLAMP 9.0 software (Molecular Devices) at a
sampling rate of 1 kHz. Data were captured with a Pentium III
personal computer (Research Machines, Abingdon, Oxfordshire,
UK). Single-channel current amplitudes were calculated from idealized traces of at least 60 s in duration using the 50% threshold
method with events lasting for ⬍6.664 ms [2 times rise time for a 100
Hz (⫺3 db) low pass filter] being excluded from analysis. In Fig. 7,
outward channel currents are shown as upward deflections. NP0 was
calculated using eq. 2:
NP0 ⫽
冘
(Onn)
T
(2)
Results
Characterization of Ca2ⴙ-Activated Clⴚ Currents in
Mouse Portal Vein Myocytes. Although sustained IClCa
evoked by pipette solutions of known free [Ca2⫹] have been
characterized extensively in rabbit pulmonary artery, coronary artery, and portal vein (Greenwood et al., 2001, 2004;
Ledoux et al., 2003, 2005; Angermann et al., 2006), our previous study in mouse portal vein (Britton et al., 2002) only
looked at a limited range of [Ca2⫹]. Figure 1A shows typical
families of IClCa recorded from different mouse PV smooth
muscle cells dialyzed with a pipette solution containing 20,
100, 250, 500, or 1000 nM Ca2⫹. These experiments clearly
show that as the intracellular Ca2⫹ was raised, the current at
a holding potential of ⫺60 mV increased consistent with the
tonic activation of Cl⫺ channels. Depolarization generated
distinctive voltage-dependent kinetics that are of IClCa elicited by this technique (Arreola et al., 1996; Nilius et al., 1997;
Kuruma and Hartzell, 2000; Greenwood et al., 2001, 2004;
Ledoux et al., 2003, 2005; Angermann et al., 2006). Ilate
measured at the end of a depolarizing step (Fig. 1B), composed by the sum of an instantaneous (Iinitial) and a timedependent current component, exhibited strong outward
rectification, a property previously ascribed to voltage-dependent gating. The slow inward tail current (Itail) detected upon
repolarization to ⫺60 mV was consistent with voltage-dependent deactivation. These currents were maintained for at
least 10 min after membrane rupture without any significant
change in amplitude (data not shown). The evoked current
reversed at ⫺3.1 ⫾ 2 mV (n ⫽ 14) near the theoretical
equilibrium potential for Cl⫺ (⫺5 mV), and the reversal potential was shifted to more negative potentials (⫺44.5 ⫾ 4
mV; n ⫽ 9) upon replacement of external NaCl with NaSCN.
This gave a PSCN/PCl of 5.5 ⫾ 0.45 (n ⫽ 9) that was identical
to that recorded in other smooth muscle cell types (e.g.,
Greenwood and Large, 1999; Piper et al., 2002). These data
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
where On is time spent at the open level; n is the number of observed
channels in the patch, and T is total recording time. Data were
analyzed using associated Clampfit software (Molecular Devices),
whereas figures were produced using Microcal Origin 6.1 or 7.5
(Northampton, MA). All data are means ⫾ S.E.M. taken from at least
three animals, whereas statistical tests were performed using paired
Student’s t test. NS1619 and NFA were purchased from SigmaAldrich (St. Louis, MO). Isopimaric acid was obtained from Alomone
Labs Ltd. (Jerusalem, Israel).
allied to the findings of Britton et al. (2002) confirmed that
the channel underlying IClCa in mouse portal vein myocytes
exhibits very similar biophysical properties as Ca2⫹-dependent Cl⫺ channels in other vascular myocytes (Greenwood et
al., 2001, 2004; Ledoux et al., 2003, 2005; Angermann et al.,
2006).
The Effect of NS1619 on IClCa. NS1619 is a benzimidazole compound that stimulates Ca2⫹-activated potassium
(K⫹) currents (IBKCa) in smooth muscle preparations at concentrations between 3 and 100 ␮M (Olesen et al., 1994;
Holland et al., 1996; Huang et al., 1997). Application of 30
␮M NS1619, a concentration shown to produce a marked
stimulation of IBKCa in smooth muscle (Olesen et al., 1994;
Holland et al., 1996; Huang et al., 1997), enhanced the amplitude of IClCa considerably within 3 min of cell dialysis (Fig.
2A) that was manifest as a greater increase in the amplitude
of IClCa at the end of the test step. For IClCa activated by 250
nM [Ca2⫹]i, 30 ␮M NS1619 increased the amplitude of Iinitial
by 23% from 39.5 ⫾ 5 to 48.6 ⫾ 5 pA (n ⫽ 14, p ⫽ 0.04, paired
Student’s t test), whereas the amplitude of Ilate was increased
93% by this compound from 104 ⫾ 18 to 200 ⫾ 36 pA (p ⬍
0.0001). Application of the appropriate vehicle had no significant effect on IClCa (n ⫽ 3, data not shown). The effects of
NS1619 were reversed significantly upon washout of the
agent (Fig. 2A) and were concentration-dependent, with 300
nM and 3 ␮M NS1619 producing an increase in the amplitude of the late current at ⫹70 mV of 8 ⫾ 6% (n ⫽ 4) and 29 ⫾
9% (n ⫽ 5), respectively. Moreover, 30 ␮M NS1619 had no
effect on the negligible current evoked with pipette solutions
containing 20 nM [Ca2⫹] (n ⫽ 3). NS1619 had no effect on the
time-dependent kinetics exhibited by IClCa (compare panels
Aii and Bii in Fig. 3). Moreover, the increase in current
amplitude seen in the presence of NS1619 was not due to the
de novo activation of a conductance distinct from IClCa because the current recorded in the presence of 30 ␮M NS1619
displayed a reversal potential that was close to the equilibrium potential for chloride ions and not significantly different from that determined in the absence of drug (⬃⫺1 mV;
Fig. 2B). Furthermore, the reversal potential of the current
recorded in the presence of NS1619 was shifted in a similar
manner to control IClCa (see description above; also Britton et
al., 2002) by replacement of the external Cl⫺ with SCN⫺
(Erev, ⫺43 ⫾ 13 mV, n ⫽ 4; data not shown). IpA is a pimarine
compound structurally unrelated to NS1619 that also augments IBKCa via an interaction with the pore forming subunit
(Imaizumi et al., 2002). IpA had similar effects on IClCa as
NS1619, and at ⫹80 mV, 3 ␮M IpA increased Ilate evoked by
250 nM Ca2⫹ from 3.4 ⫾ 1 to 7.7 ⫾ 5 pA/pF (p ⬍ 0.001; paired
Student’s t test). Similar to NS1619, IpA did not affect the
reversal potential of the evoked current (⫺3 ⫾ 0.3 and
⫺3.3 ⫾ 0.4 mV, n ⫽ 4, in the absence and presence of IpA)
and had no effect on any of the kinetics of IClCa (e.g., at ⫹80
mV, the mean time constant for current development was
542 ⫾ 46 and 505 ⫾ 52 ms in the absence and presence of
IpA, n ⫽ 9, p ⫽ 0.3). Overall, these data show that two
structurally unrelated activators of BKCa enhanced IClCa in
murine portal vein myocytes.
Effect of Niflumic Acid on IClCa Enhanced by NS1619.
Similar to studies on rabbit pulmonary artery and coronary
artery myocytes that revealed a paradoxical stimulatory effect of the chloride channel blocker NFA on IClCa (for structure, see Greenwood and Large, 1995; Piper et al., 2002;
Modulation of Calcium-Activated Chloride Channels
1079
Fig. 3. Effect of niflumic acid on IClCa in the
absence or presence of NS1619 in murine portal
vein myocytes. Ai, representative experiment
showing the effect of 100 ␮M NFA on IClCa
evoked by dialysis with 500 nM free [Ca2⫹]. Aii,
effect of NFA on the mean time constants for
activation at ⫹90 mV (rise, ⽧) and deactivation
at ⫺80 mV (decay, ⽧). Bi, effect of 100 ␮M NFA
on IClCa augmented by 30 ␮M NS1619. Dotted
line, control currents. Bii, effect of NFA on the
mean rate of activation at ⫹90 mV (rise t) and
mean time constant for deactivation at ⫺80 mV
(decay t) elicited in control and after exposure to
NS1619. All data are the mean ⫾ S.E.M. of six to
nine cells.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Fig. 4. Effect of NS1619 on the Ca2⫹ sensitivity of IClCa in murine portal vein myocytes. A, six graphs summarizing the effects of NS1619 on the [Ca2⫹]I
dependence of IClCa conductance measured at membrane potentials ranging from ⫺100 to ⫹80 mV. All data are the means ⫾ S.E.M. of four to seven
cells. The lines passing through the control and NS1619 data points at ⫺100, ⫺80, ⫺60, and ⫹80 mV are B-spline fits calculated by Origin software.
For the remaining two graphs at ⫹40 and ⫹60 mV, the lines are least-squares fits to a logistic function (with error bars taken into account for
weighting) of the following form: y ⫽ (A1 ⫺ A2)/(1 ⫹ (x/x0)p) ⫹ A2, where y represents IClCa conductance (nanosiemens), A1 and A2 are, respectively, the
maximal and minimal conductance levels (nanosiemens), x is [Ca2⫹]i (nanomolar), x0 is the [Ca2⫹]i yielding half-maximal IClCa conductance
(nanomolar; apparent Kd for Ca2⫹), and p is the power factor (index of steepness of the relationship). B, bar graph reporting the effect of NS1619 on
the apparent Kd ⫾ fitting error scaled to the square root of ␹2 for Ca2⫹ at ⫹40 and ⫹60 mV estimated from the analyses of the data sets displayed
in A. The calculated errors for the NS1619 bars are within the thickness of bar lines.
Ledoux et al., 2005), a supramaximal concentration of NFA
(100 ␮M) inhibited Ilate at ⫹80 mV in murine PV myocytes by
32 ⫾ 11% (Fig. 3Ai; n ⫽ 6) that was associated with a slowing
of the rate of current development (␶ ⫽ 296 ⫾ 37 ms in control
and 717 ⫾ 74 ms with NFA; p ⬍ 0.01; see Fig. 3Aii), which
was reversible upon washout. In addition, NFA increased the
current at the holding potential of ⫺60 mV by 58 ⫾ 5 pA and
slowed significantly the decay of Itail (Fig. 3Aii). In the presence of 30 ␮M NS1619, application of 100 ␮M NFA inhibited
Ilate by 52 ⫾ 9% (Fig. 3Bi), and similar to control IClCa, NFA
prolonged the development of current at ⫹80 mV from 256 ⫾
23 to 415 ⫾ 138 ms (n ⫽ 4; p ⫽ 0.03) and increased the decay
1080
Saleh et al.
Ca2⫹; e.g., at ⫹40 mV, the Kd for Ca2⫹ was 529 nM in control
and 413 nM in the presence of 30 ␮M NS1619.
NS1619 also had a marked effect on the voltage dependence of IClCa activation (Fig. 5). NS1619 produced little
effect on IClCa conductance when the cells were dialyzed with
100 nM Ca2⫹ (Fig. 5A, top left). A significant elevation of
IClCa conductance by NS1619 with 250 nM Ca2⫹ was detectable at potentials more positive than ⫹20 mV (Fig. 5A, top
right). A more pronounced effect of NS1619 became evident
at higher [Ca2⫹]i characterized by a leftward shift of the
voltage dependence of IClCa conductance, leading to enhancement of steady-state IClCa at negative potentials (Fig. 5A,
bottom), especially with 1 ␮M Ca2⫹ (e.g., 3.4-fold increase at
⫺60 mV). All the data sets in Fig. 5A were fitted to a Boltzmann relationship to determine the values for half-maximal
activation of IClCa by voltage (V0.5). For all fits, fitting was
constrained to a maximal conductance of 11.94 nS, which was
determined by fitting the NS1619 data sets with 1 ␮M Ca2⫹
(Fig. 5A, bottom right). The V0.5 values estimated in control
and in the presence of NS1619 were plotted as a function of
[Ca2⫹]i in Fig. 5B. As previously shown for IClCa recorded
from rabbit pulmonary artery myocytes (Angermann et al.,
2006), V0.5 declined exponentially as a function of [Ca2⫹]i in
control. V0.5 decreased significantly with NS1619 for [Ca2⫹]i
between 250 and 1000 nM. The absolute magnitude of the
NS1619-induced shift of V0.5 increased from 34 mV with 250
nM [Ca2⫹]i to 84 mV with 1000 nM [Ca2⫹]i (Fig. 5C), indi-
Fig. 5. Effect of NS1619 on the voltage dependence of IClCa in murine portal vein myocytes. A, six graphs summarizing the effects of NS1619 on the
voltage dependence of IClCa conductance measured at the four intracellular Ca2⫹ concentrations as indicated. All data are the means ⫾ S.E.M. of four
to seven cells. All lines passing through the data points are least-squares fits to a Boltzmann function (with error bars taken into account for weighting)
of the following form: y ⫽ ((A1 ⫺ A2)/(1 ⫹ exp ((x ⫺ x0)/dx))) ⫹ A2, where y represents IClCa conductance (nanosiemens), A1 and A2 are, respectively,
the maximal and minimal conductance levels (nanosiemens), x is the step voltage (millivolts), x0 is the voltage for half-maximal activation or V0.5
(millivolts; see B), and dx is the slope factor (millivolts). As explained in the text, all data sets were fitted to the latter equation by limiting the maximal
conductance level (A1) to 12.94 nS, a value determined based on the fitting of the NS1619 group of data (open circles) measured with 1000 nM Ca2⫹
(bottom side graph). From the curve fitting analysis of the data presented in A, the V0.5 values (mean ⫾ fitting error scaled with square root of ␹2)
determined for the control (filled squares) and NS1619 (open circles) data sets were plotted as a function of [Ca2⫹]i in B. The lines are monoexponential
fits to the data points with error bars taken into account for weighting. C, graph of the shift in V0.5 induced by NS1619 as a function of [Ca2⫹]i; the
data points were calculated by a simple subtraction of the V0.5 values (control ⫺ NS1619) shown in B for each [Ca2⫹]i.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
of Itail at ⫺60 mV (p ⫽ 0.004; n ⫽ 4; Fig. 3Bii). These data
show that the augmentation of IClCa by NS1619 does not
affect the interaction of NFA with the channel. It is important to stress that in the absence of NFA, NS1619 did not
significantly alter the time constants of activation and deactivation of IClCa relative to control (compare the open bars in
Aii and Bii).
Does the NS1619-Induced Increase in IClCa Involve a
Change in Ca2ⴙ or Voltage Dependence? As shown earlier, activation of IClCa is reliant upon the level of free [Ca2⫹]
in the pipette solution, a process that is influenced by membrane potential (Angermann et al., 2006). We thus examined
whether NS1619 might exert its stimulatory effect by altering the Ca2⫹ and/or voltage dependence of IClCa. Figure 4A
presents the results of our analysis of the Ca2⫹ dependence of
the steady-state chord conductance of IClCa at membrane
potentials spanning the negative and positive membrane potential ranges. As three of the six panels clearly show, it was
not possible to determine an apparent Kd for Ca2⫹ at negative potentials because the conductance displayed no sign of
saturation at elevated [Ca2⫹]i in the absence or presence of
NS1619. However, both the control and NS1619 data sets at
potentials ⱖ ⫹40 mV were successfully fitted to a logistic
function yielding apparent Kd values for Ca2⫹ that declined
linearly with membrane potential in both groups between
⫹40 and ⫹100 mV (Fig. 4B). The graph in Fig. 4B shows that
NS1619 increased the sensitivity of IClCa to intracellular
Modulation of Calcium-Activated Chloride Channels
relationship that reversed close to ⫺50 mV (a cesium-aspartate-based bathing solution was used that set the equilibrium potential for chloride ions at approximately ⫺50 mV)
with slope conductance values calculated to be 1.8 ⫾ 0.3 and
3.1 ⫾ 0.2 pS (n ⫽ 6, Fig. 7A). These values are similar to
single IClCa characterized by Piper and Large (2003), who
suggested that the larger conductance is the fully open channel, whereas the smaller is a subconductance state. In control
condition, the open time distribution was fitted to a double
exponential function with mean open times of 4.8 ⫾ 2 and
15 ⫾ 6 ms (n ⫽ 4). Application of 30 ␮M NS1619 resulted in
an immediate and reversible effect on channel activity causing a significant increase in the frequency of channel opening
over an identical period of time (Fig. 7, B and C). due to the
presence of multiple channels in the patch and the very high
channel activity elicited by NS1619, kinetic analysis could
not be performed in the presence of this agent. With 500 nM
[Ca2⫹]i, NP0 was significantly enhanced from 0.271 ⫾ 0.14 in
control to 1.52 ⫾ 0.38 with 30 ␮M NS1619 (Fig. 7D).
Discussion
This study shows for the first time that two structurally
distinct activators of BKCa, NS1619 and isopimaric acid,
augmented sustained IClCa in vascular smooth muscle cells.
As for IClCa recorded in the absence of drug, the current
enhanced by NS1619 reversed near the equilibrium potential
for Cl⫺, its reversal potential, was shifted by over ⫺40 mV by
replacement of extracellular Cl⫺ with SCN⫺, was blocked by
the putative IClCa blocker niflumic acid, and exhibited a
unitary conductance consistent with that reported for single
Ca2⫹-activated Cl⫺ channels (ClCa) recorded in many types of
vascular smooth muscles (1–3 pS; Van Renterghem and Lazdunski, 1993; Hirakawa et al., 1999; Piper and Large, 2003).
A careful analysis of the biophysical characteristics of IClCa
revealed that NS1619 enhanced IClCa by increasing the sensitivity of the channels to Ca2⫹ and shifting their voltage
Fig. 6. Effect of NS1619 on IClCa recorded in
rabbit pulmonary artery myocytes. A, representative trace of IClCa evoked by 500 nM [Ca2⫹] and
recorded at different potentials in the absence
and presence of 30 ␮M NS1619. Cells were held
at ⫺60 mV and depolarized to ⫹80 mV for 750
ms followed by repolarization to ⫺80 mV for 1 s.
B, 100 ␮M NFA had qualitatively similar effects
on IClCa as it has on control IClCa. C, mean kinetics of IClCa at ⫹80 and ⫺80 mV in the absence
(open bars) and presence (filled bars) of 30 ␮M
NS1619. Mean of six cells ⫾ S.E.M.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
cating that the effects of the BKCa opener on the voltage
dependence of IClCa are Ca2⫹-dependent.
Effects of NS1619 in Rabbit Pulmonary Artery Myocytes. IClCa generated in murine PV myocytes were qualitatively similar to IClCa evoked by this technique in rabbit
pulmonary artery, coronary artery, and PV myocytes (Greenwood et al., 2001). However, it is possible that the stimulatory effect of NS1619 was a “quirk” of the murine PV myocytes. Consequently, experiments were undertaken to
determine whether NS1619 also augmented IClCa in rabbit
pulmonary artery myocytes, a cell type where IClCa has been
studied extensively (Greenwood et al., 2001, 2004; Angermann et al., 2006). NS1619 (30 ␮M) had similar effects on
IClCa evoked in pulmonary artery myocytes (Fig. 6A). The
amplitude of Ilate was increased 63 ⫾ 8% in eight cells with
no significant effect on the kinetics of IClCa either at ⫹90 or
⫺80 mV (Fig. 6C). The augmented IClCa recorded in the
presence of NS1619 was modulated by 100 ␮M NFA in a
manner identical to control IClCa recorded in the absence of
NS1619 (Fig. 6B). These data show that the enhancement of
IClCa by NS1619 is a characteristic of vascular IClCa and is
not unique to the mouse PV.
Properties of Single Ca2ⴙ-Activated Clⴚ Channels in
Inside-out Patches. IClCa was investigated in the inside-out
configuration of the patch-clamp technique so that the
[Ca2⫹]i could be manipulated, and the reagents could be
applied directly onto the intracellular surface of the membrane. Piper and Large (2003) have previously conducted a
thorough analysis on the single-channel IClCa in this particular preparation; therefore, extensive characterization was
not deemed necessary. At ⫹80 mV in low-Ca2⫹ solution (14
nM), no channel activity was present (Fig. 7B), but the rapid
addition of 500 nM Ca2⫹ evoked an outwardly directed single-channel current. This Ca2⫹-activated channel opened to
two levels of approximately 0.2 and 0.4 pA (see Fig. 7, Bii and
Ci), which may represent either two channels in the patch or
two conductance levels. Both channels displayed a linear I-V
1081
1082
Saleh et al.
dependence toward negative potentials that resulted in an
increase in open probability and/or number of available channels. These findings, allied to the observation that Cl⫺ channel blockers stimulate BKCa, show that there is a similarity
in the pharmacology of calcium-activated chloride channel
and large-conductance calcium-dependent potassium channel encoded by Slo1.
Activators of BKCa Stimulate IClCa. Extensive studies
on the effect of global dephosphorylation on IClCa in rabbit
pulmonary artery myocytes revealed that augmentation of
IClCa produced by prohibiting phosphorylation by intracellular dialysis with the nonhydrolyzable ATP analog adenylyl5⬘-imidodiphosphate was mediated by a marked negative
shift in the voltage dependence of activation (Angermann et
al., 2006). It is possible that NS1619 or IpA enhanced IClCa by
blocking CaMKII (Greenwood et al., 2001) and/or by stimulating calcineurin (Ledoux et al., 2003; Greenwood et al.,
2004), which have been shown to induce such shifts. However, this seems unlikely because alterations in the phosphorylation status of the channel or regulatory subunit were
accompanied by marked changes in activation and deactivation kinetics of IClCa, whereas those produced by NS1619 or
IpA were not. Future studies will be undertaken to ascertain
how phosphorylation affects the stimulatory action of
NS1619 and IpA.
NS1619 (0.3–30 ␮M) and IpA (3 ␮M) enhanced IClCa at
concentrations that increase BKCa markedly (Olesen et al.,
1994; Imaizumi et al., 2002). Because IClCa in the present
study was evoked without reliance upon other mechanisms
such as the opening of Ca2⫹ channels or Ca2⫹ release processes the stimulatory effect of NS1619 and IpA reflected an
augmentation of the Cl⫺ channel activity as opposed to a
perturbation of Ca2⫹ homeostatic mechanisms. Analysis of
the effect of NS1619 on the intrinsic Ca2⫹ or voltage sensitivity of IClCa (described extensively by Angermann et al.,
2006) showed that NS1619 reduced the apparent Kd for Ca2⫹
at positive potentials and caused a negative shift in the
voltage dependence of activation, resulting in basal activation of the channels at negative potentials in the presence of
1 ␮M Ca2⫹. Furthermore, other experiments confirmed that
application of NS1619 to excised patches reversibly increased
NP0 of a low conductance (⬃3 pS) anion channel whose
activation had an absolute requirement for an elevated Ca2⫹
concentration facing the cytoplasmic side of the membrane
patch. Consistent with our whole-cell data, the channel stimulated by NS1619 had similar characteristics to those of ClCa
described in this preparation (Piper and Large, 2003) and
thus was not due to the activation of an unidentified silent
channel. Because a detailed kinetic analysis could not be
performed due to the presence of numerous channels in all
patches tested, it was not possible to conclude unequivocally
whether NS1619 exerted its effects on single ClCa by increasing their open probability, by recruiting silent channels, or
both. Similar to Piper and Large (2003), we identified two
conductance levels in our single-channel experiments, and
although not analyzed in detail, our data suggest that
NS1619 did not produce a shift in the proportion of the two
conductance levels. Clearly, more experiments are necessary
to address this question, in particular by examining the Ca2⫹
and voltage dependence of single ClCa.
The ability of all the BKCa modulators to affect IClCa could
simply be a pluripotent effect of “dirty drugs.” However, the
likelihood of two specific activators of BKCa having nonspecific effects seems remote. This leaves four plausible interpretations of our data. Firstly, the enhancement of the
evoked current by NS1619 and IpA could have been due to
the de novo activation of IBKCa. However, the fact that the
current in the presence and absence of either activator reversed close to the theoretical Cl⫺ equilibrium potential,
which was shifted by the same extent upon replacement of
the external Cl⫺ by the more permeable anion thiocyanate,
suggests that this scenario is unlikely. Any contribution to
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Fig. 7. The effect of NS1619 on
unitary IClCa in smooth muscle
cells from the rabbit pulmonary
artery. A, I-V relationship showing
the two conductance levels that
were predominantly seen in the inside-out configuration with 500
nM [Ca2⫹]i. The mean data were
fitted with linear relationships
yielding the slope conductance
values shown. Bi, long-term trace
from a patch being held at ⫹80
mV exposed initially to 14 nM
[Ca2⫹]i and subsequently to 500
nM [Ca2⫹]i in the presence and absence of 30 ␮M NS1619, all applied
to the internal surface of the membrane. No channel activity was
seen with 14 nM [Ca2⫹]i, and this
is highlighted in Bii, trace 1, which
shows the channel activity of the
corresponding long-term trace on a
shorter time scale. Bii, traces 2
and 3, correspond to channel activity with 500 nM [Ca2⫹]i and 30 ␮M
NS1619, respectively. C, two amplitude histograms displaying the
frequency of channel openings
within a 2-min period before (Ci)
and after (Cii) the application of 30
␮M NS1619. D, bar graph, effect of
NS1619 on channel NP0.
Modulation of Calcium-Activated Chloride Channels
revealed no homology of mouse Slo1 with either CLCA or
Bestrophin genes when aligned specifically. Recently, Suzuki
and Mizuno (2004) identified genes called Tweety (ttyh1–3),
whose products have comparable structure with BKCa and
encode for a large-conductance Cl⫺ channel activated by an
increase in [Ca2⫹] with an EC50 of 2 ␮M. Although the
biophysical properties of the currents generated by expression of ttyh3 were not similar to native IClCa in smooth
muscle cells, this paper highlights that putative Cl⫺ channel
correlates can have considerable homology with K⫹ channels.
It is likely that the protein forming the ClCa is a multimeric
complex that may incorporate a number of the proteins described above. Interestingly, coexpression of the ␤ subunit
that associates with mSlo1 expression products to form native BKCa has been shown to increase the Ca2⫹ sensitivity of
mCLCA1 (Greenwood et al., 2002). Overall, the data of the
present study suggest that determination of the elusive nature of the Ca2⫹-activated Cl⫺ channel may lie with previously unconsidered protein interactions.
References
Arreola J, Melvin JE, and Begenisich T (1996) Activation of calcium-dependent
chloride channels in rat parotid acinar cells. J Gen Physiol 108:35– 47.
Angermann JE, Sanguinetti AR, Kenyon JL, Leblanc N, and Greenwood IA (2006)
Mechanism of the inhibition of Ca2⫹-activated-chloride currents by phosphorylation in pulmonary arterial smooth muscle cells. J Gen Physiol 128:73– 87.
Bhattacharjee A and Kaczmarek LK (2005) For K⫹ channels, Na⫹ is the new Ca2⫹.
Trends Neurosci 28:422– 428.
Britton FC, Ohya S, Horowitz B, and Greenwood IA (2002) Comparison of the
properties of CLCA1 generated currents and ICl(Ca) in murine portal vein smooth
muscle cells. J Physiol 539:107–117.
Butler A, Tsunoda S, McCobb DP, Wei A, and Salkoff L (1993) MSlo, a complex
mouse gene encoding “maxi” calcium-activated potassium channels. Science 261:
221–224.
Gibson A, Lewis AP, Affleck K, Aitken AJ, Meldrum E, and Thomson N (2005)
hCLCA1 and mCLCA3 are secreted non-integral membrane proteins and therefore
are not ion channels. J Biol Chem 280:27205–27212.
Greenwood IA and Large WA (1995) Comparison of the effects of fenamates on
Ca-activated chloride and potassium currents in rabbit portal vein smooth muscle
cells. Br J Pharmacol 116:2939 –2948.
Greenwood IA and Large WA (1999) Modulation of Ca2⫹-activated Cl⫺ currents in
rabbit portal vein smooth muscle cells by external anions. J Physiol 516:365–376.
Greenwood IA, Ledoux J, and Leblanc N (2001) Differential regulation of Ca2⫹activated Cl⫺ currents in rabbit arterial and portal vein smooth muscle cells by
Ca2⫹-calmodulin-dependent kinase. J Physiol 534:395– 408.
Greenwood IA, Ledoux J, Sanguinetti A, Perrino BA, and Leblanc N (2004) Calcineurin A␣ but not A␤ augments ICl(Ca) in rabbit pulmonary artery smooth muscle
cells. J Biol Chem 279:38830 –38837.
Greenwood IA, Miller LJ, Ohya S, and Horowitz B (2002) The large conductance
potassium channel ␤ subunit can interact with and modulate the functional
properties of a calcium-activated chloride channel CLCA1. J Biol Chem 277:
22119 –22122.
Hartzell C, Putzier I, and Arreola J (2005) Calcium-activated chloride channels.
Annu Rev Physiol 67:719 –758.
Holland M, Langton PD, Standen NB, and Boyle JP (1996) Effects of the BKCa
channel activator, NS1619, on rat cerebral artery smooth muscle. Br J Pharmacol
117:119 –129.
Hirakawa Y, Gericke M, Cohen RA, and Bolotina VM (1999) Ca2⫹-dependent Cl⫺
channels in mouse and rabbit aortic smooth muscle cells: regulation by intracellular Ca2⫹ and NO. Am J Physiol 277:H1732–H1744.
Huang Y, Lau CW, and Ho IH (1997) NS 1619 Activates Ca2⫹-activated K⫹ currents
in rat vas deferens. Eur J Pharmacol 325:21–27.
Imaizumi Y, Sakamoto K, Yamada A, Hotta A, Ohya S, Muraki K, Uchiyama M, and
Ohwada T (2002) Molecular basis of pimarane compounds as novel activators of
large-conductance Ca2⫹-activated K⫹ channel ␣-subunit. Mol Pharmacol 62:836 –
846.
Joiner WJ, Tang MD, Wang LY, Dworetzky SI, Boissard CG, Gan L, Gribkoff VK,
and Kaczmarek LK (1998) Formation of intermediate-conductance calciumactivated potassium channels by interaction of Slack and Slo subunits. Nature
Neurosci 1:462– 469.
Kuruma A and Hartzell HC (2000) Bimodal control of Ca2⫹-activated Cl⫺ channel by
different Ca2⫹ signals. J Gen Physiol 115:59 – 80.
Large WA and Wang Q (1996) Characteristics and physiological role of the Ca2⫹activated Cl⫺ conductance in smooth muscle. Am J Physiol 271:C435–C454.
Leblanc N, Ledoux J, Saleh S, Sanguinetti A, Angermann J, O’Driscoll K, Britton F,
Perrino BA, and Greenwood IA (2005) Regulation of calcium-activated chloride
channels in smooth muscle cells: a complex picture is emerging. Can J Physiol
Pharmacol 83:541–556.
Ledoux J, Greenwood IA, and Leblanc N (2005) Dynamics of Ca2⫹-dependent Cl⫺
channel modulation by niflumic acid in rabbit coronary arterial myocytes. Mol
Pharmacol 67:163–173.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
the gross current due to BKCa activation would be negligible
due to the presence of TEA in the internal and external
solutions, lack of external K⫹, and the presence of Cs⫹ instead of K⫹ in the internal solution. Consequently, the stimulatory effect produced by NS1619 or IpA was the result of a
modulation of the activated Cl⫺ conductance and not by
unmasking a quiescent BKCa or another conductance. Secondly, IClCa recorded in K⫹-free conditions in the present
study and similar previous investigations represent an abstract scenario with Cl⫺ ions permeating an extant BKCa.
There are many splice variants of the Slo genes (Butler et al.,
1993; Tseng-Crank et al., 1994), but none of them correlates
with an anion permeable pore. Moreover, the anion permeability of the sustained IClCa in various vascular smooth
muscle cells (Greenwood et al., 2001; Piper et al., 2002;
present study) is identical to transient IClCa occurring either
spontaneously or following the influx of Ca2⫹ through voltage-dependent channels (e.g., Greenwood and Large, 1999).
Furthermore, spontaneously occurring IClCa have been recorded in K⫹-containing conditions contemporaneously with
spontaneous K⫹ currents carried by BKCa (e.g., Wang et al.,
1992; Large and Wang, 1996; ZhuGe et al., 1998; present
study; data not shown). A final possibility is that Slo1 somehow interacts physically with neighboring ClCa. However,
the most prosaic and simplest hypotheses that can be derived
from the data of the present study, allied to the findings that
CCBs activate BKCa, is that there are some similarities in the
proteins underlying native IClCa and those that are encoded
by Slo1, which underpins the similarity in the pharmacology
of IClCa and BKCa.
Molecular Identity of the Ca2ⴙ-Activated Clⴚ Channel. To date two families of genes (CLCA and bestrophin)
have been postulated to underlie the Ca2⫹-dependent Cl⫺
conductance, but the role of CLCA gene products as ion
channels has been repudiated on the strength of the poor
membrane topology and immunocytochemical evidence,
whereas some of these proteins are speculated to exist as
secretory products (Gibson et al., 2005; Loewen and Forsyth,
2005). Bestrophin genes are expressed in vascular smooth
muscle cells (Leblanc et al., 2005), and the expression of
Bestrophin genes in HEK-293 cells produces Ca2⫹-sensitive
Cl⫺ conductances (Sun et al., 2002) at physiological [Ca2⫹]
(Qu et al., 2004). Moreover, mutations of specific amino acids
within the putative pore region have been shown to alter ion
conductance proving that these proteins do form anion channels (Qu et al., 2004). However, there are considerable differences between native IClCa and currents produced by Bestrophin expression in mammalian cell lines, and we do not
know if BKCa activators enhance Cl⫺ currents produced by
the heterologous expression of Bestrophin genes.
The crossover in channel pharmacology suggest that gene
products of Slo1 or other members of the Slo family of genes
(Slick, Slo2.1; Slack, Slo2.2) could also be considered as molecular candidates for the channel underlying IClCa. Channels encoded by Slo1 are K⫹ channels so cannot underlie
IClCa per se, and mouse Slo2.1 and Slo2.2 both encode for
Na⫹ activated K⫹ channels whose activity is influenced by
intracellular [Cl⫺] (Bhattacharjee and Kaczmarek, 2005).
Moreover, specific alignment of all possible combinations of
mouse CLCA and Bestrophin genes with mouse slo1 revealed
no homology using BLAST searches of the NCBI protein
database. BLAST searches of the NCBI protein database also
1083
1084
Saleh et al.
Ledoux J, Greenwood IA, Villeneuve LR, and Leblanc N (2003) Modulation of
Ca2⫹-dependent Cl⫺ channels by calcineurin in rabbit coronary arterial myocytes.
J Physiol 552:701–714.
Loewen ME and Forsyth GW (2005) Structure and function of CLCA proteins.
Physiol Rev 85:1061–1092.
Nilius B, Prenen J, Voets T, van Den Bremt K, Eggermont J, and Droogmans G
(1997) Kinetic and pharmacological properties of the calcium-activated chloride
current in macrovascular endothelial cells. Cell Calcium 22:53– 63.
Olesen SP, Much E, Moldt P, and Drejer J (1994) Selective activation of Ca2⫹dependent K⫹ channels by novel benzimidazolone. Eur J Pharmacol 251:53–59.
Ottolia M and Toro L (1994) Potentiation of large conductance KCa channels by
niflumic, flufenamic, and mefenamic acids. Biophys J 67:2272–2279.
Piper AS, Greenwood IA, and Large WA (2002) Dual effect of blocking agents on
Ca2⫹-activated Cl⫺ currents in rabbit pulmonary artery smooth muscle cells.
J Physiol 539:119 –131.
Piper AS and Greenwood IA (2003) Anomalous effect of anthracene-9-carboxylic acid
on calcium-activated chloride currents in rabbit pulmonary artery smooth muscle
cells. Br J Pharmacol 138:31–38.
Piper AS and Large WA (2003) Multiple conductance states of single Ca2⫹-activated
Cl⫺ channels in rabbit pulmonary artery smooth muscle cells. J Physiol 547:181–196.
Qu Z, Fischmeister R, and Hartzell C (2004) Mouse bestrophin-2 is a bona fide Cl⫺
channel: identification of a residue important in anion binding and conduction.
J Gen Physiol 123:327–340.
Saleh S and Greenwood IA (2005) Activation of chloride currents in murine portal
vein smooth muscle cells by membrane depolarization involves intracellular calcium release. Am J Physiol 288:C122–C131.
Sun H, Tsunenari T, Yau KW, and Nathans J (2002) The vitelliform macular
dystrophy protein defines a new family of chloride channels. Proc Natl Acad Sci
USA 99:4008 – 4013.
Suzuki M and Mizuno A (2004) A novel human Cl⫺ channel family related to
Drosophila flightless locus. J Biol Chem 279:22461–22468.
Toma C, Greenwood IA, Helliwell RM, and Large WA (1996) Activation of potassium
currents by inhibitors of calcium-activated chloride conductance in rabbit portal
vein smooth muscle cells. Br J Pharmacol 118:513–520.
Tseng-Crank J, Foster CD, Krause JD, Mertz R, Godinot N, Dichiara TJ, and
Reinhart PH (1994) Cloning, expression, and distribution of functionally distinct
Ca2⫹-activated K⫹ channel isoforms from human brain. Neuron 13:1315–1330.
Van Renterghem C and Lazdunski M (1993) Endothelin and vasopressin activate low
conductance chloride channels in aortic smooth muscle cells. Pflugers Arch 425:
156 –163.
Wang Q, Hogg RC, and Large WA (1992) Properties of spontaneous inward currents
recorded in smooth muscle cells isolated from the rabbit portal vein. J Physiol
451:525–537.
ZhuGe RH, Sims SM, Tuft RA, Fogarty KE, and Walsh JV Jr (1998) Ca2⫹ sparks
activate K⫹ and Cl⫺ channels, resulting in spontaneous transient currents in
guinea-pig tracheal myocytes. J Physiol 513:711–718.
Address correspondence to: Dr. Iain A. Greenwood, Ion Channels and Cell
Signaling Research Centre, Division of Basic Medical Sciences, St. George’s,
University of London, SW17 0RE London, UK. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017