Full text of DOI:10.1111/j.1469-7793.2000.00521.x

1188
Journal of Physiology (2000), 528.3, pp.521–537
521
Membrane currents in cultured human intestinal smooth
muscle cells
A. V. Zholos, C. J. Fenech, S. A. Prestwich and T. B. Bolton
Department of Pharmacology and Clinical Pharmacology, St George’s Hospital Medical
School, London SW17 ORE, UK
(Received 1 June 2000; accepted 19 July 2000)
1. Using whole-cell patch-clamp recording techniques, we have examined voltage-gated ion
currents in a cultured human intestinal smooth muscle cell line (HISM). Experiments were
performed at room temperature on cells after passages 16 and 17.
2. Two major components of the whole-cell current were a tetraethylammonium-sensitive
(IC50 = 9 m
M
), iberiotoxin-resistant, delayed rectifier K+ current and a Na+ current inhibited
by tetrodotoxin (IC50 ≈ 1 00 n
M
). No measurable inward current via voltage-gated Ca2+
channels could be detected in these cells even with 10 m
Ca2+ or Ba2+ in the external solution.
M
No current attributable to calcium-activated K+ channels was found and no cationic current in
response to muscarinic receptor activation was present.
3. In divalent cation-free external solution two additional currents were activated: an inwardly
rectifying hyperpolarization-activated current, IHA, and a depolarization-activated current,
IDA.
4. IHA and IDA could be carried by several monovalent cations; the sizes of currents in descending
order were: K+ > Cs+ > Na+ for IHA and Na+ > K+ >> C s + for IDA. IHA was activated and
deactivated instantaneously and showed no inactivation whereas IDA was activated,
inactivated and deactivated within tens of milliseconds. These currents were inhibited by
external calcium with an IC50 of 0.3 µ
M
for IDA and an IC50 of 20 µ for IHA.
M
5. Cyclopiazonic acid (CPA) induced an outward, but not an inward current. SK&F 96365, a
blocker of store-operated Ca2+ channels, suppressed IDA with a half-maximal inhibitory
concentration of 9 µ
and La3+ strongly suppressed IDA at 1 and 10 µ
blocking IHA (complete inhibition required a concentration of 100 µ
10–100 µ evoked about a 3-fold increase in IHA amplitude and completely abolished IDA.
M
but was ineffective in inhibiting IHA at concentrations up to 100 µ
, respectively and were less effective in
for both). Carbachol at
M
. Gd3+
M
M
M
6. We conclude that IHA and IDA are Ca2+-blockable cationic currents with different ion selectivity
profiles that are carried by different channels. IDA shows novel voltage-dependent properties
for a cationic current.
It is well known that several distinct cellular phenotypes smooth muscle cells also may retain distinct molecular
may exist during normal smooth muscle ontogenesis; in phenotypes which, depending on the initial cell density,
contrast to skeletal and cardiac muscle cells, which exhibit substrate specificity and serum supplementation, have
much more restricted cellular plasticity, mature smooth been suggested to be identical to those in vivo
muscle cells can readily dedifferentiate from the (Brittingham et al. 1998). It thus appears that cultured
contractile phenotypic state to a non-contractile (highly cells recapitulate a significant part of in vivo smooth
proliferative, migratory and synthetic) phenotype where muscle ontogenesis. Substantial electrophysiological
synthesis of a connective tissue matrix and proliferation changes possibly relevant to the proliferation process
occur (for reviews see Chamley-Campbell et al. 1979; have also been described (e.g. Snetkov et al. 1996).
Owens, 1995), thus reverting to a more primitive
In the present study we examined the properties of
phenotype in culture. This process involves considerable
voltage-dependent inward and outward currents in
morphological, electrophysiological and biochemical
cultured human intestinal smooth muscle (HISM) cells
changes and is thought to be crucial for normal smooth
(American Type Culture Collection, ATCC no.
muscle myogenesis as well as for pathogenesis. It has
1692ªCRL). This established cell line is widely used in
recently been shown that cultured gastrointestinal
biochemical and molecular biology research but data

J. Physiol 528.3
522
A. V. Zholos, C. J. Fenech, S. A. Prestwich and T. B. Bolton
Solutions
concerning the electrophysiological properties of HISM
cells are scarce. The major inward current, which we
identified as a ‘classical’ fast, TTX-sensitive Na+ current
with properties similar to those of cells freshly dispersed
from the adult human colon (Xiong et al. 1993), was
previously erroneously referred to as Ca2+ current via
Lªtype channels despite its very rapid inactivation
kinetics (Bielefeldt et al. 1996). Also, we found two
distinct Ca2+-blockable inward cationic currents to be
present in these cells, which could be elicited in divalent
cation-free external solution. One of these, IHA, was
further potentiated by the muscarinic agonist carbachol
via muscarinic receptor/Gªprotein activation since the
effect was blocked by atropine or intracellular GDP-bS
application. The other current, which we termed IDA,
showed a pronounced U-shaped I–V relationship at
negative potentials with a peak at about _30 mV.
However, in contrast to the muscarinic cationic current
widely present in differentiated gastrointestinal smooth
muscles, IDA also showed pronounced inactivation. These
two currents also had distinct pharmacological profiles.
The basic external physiological salt solution (PSS) used to maintain
the cells before giga-seal formation consisted of (m ): NaCl 120, KCl
M
6, CaCl2 2.5, MgCl2 1.2, glucose 12 and Hepes 10, pH adjusted to 7.4
with NaOH. For INa recordings KCl was replaced by NaCl (total Na+
concentration, 130 mM) and MgCl2 was removed. Cationic currents
were measured in solutions to which CaCl2 was not added. In ion
substitution experiments NaCl was replaced with equimolar KCl,
CsCl or N-methyl-D-glucamine chloride (NMDG-Cl; pH adjusted with
KOH, CsOH or HCl, respectively). To study the dependency of the
cationic current on [Ca2+]o, extracellular calcium was strongly
buffered to 36 and 487 n
and 9 m /10 m , respectively). For higher concentrations, 250 µ
1 m CaCl2 was added to nominally divalent cation-free solution.
M
using Ca2+/EGTA mixtures (4 m
M
/10 m
M
M
M
M
or
M
Complete exchange of the external solution was achieved within
about 1s as described previously (Zholos & Bolton, 1995).
Pipettes were filled with the following solution (m
MgATP 1, creatine 5, glucose 20, Hepes 10, BAPTA 10 and CaCl2 4.6
([Ca2+]i = 1 00 n ), pH adjusted to 7.4 with CsOH (total Cs+
concentration, 124 m ). GTP (1m ) was added in some experiments
where the effects of carbachol were studied. NaCl (10 m ) was added
M): CsCl 80,
M
M
M
M
in experiments where INa was measured to define the sodium
equilibrium potential (ENa). In experiments to measure K+ currents,
the pipette solution contained (mM): KCl 130, MgATP 1, creatine 5,
glucose 20, Hepes 10 and EGTA 0.05, pH adjusted to 7.4 with NaOH.
We could not identify in HISM cells a muscarinic cationic
current with a typical U-shaped voltage dependence as
seen in all studied gastrointestinal smooth muscle cells
from various non-human mammalian species (e.g. Benham
et al. 1985; Inoue & Isenberg, 1990; Zholos & Bolton,
1994). From the above considerations it remains to be
established whether such a current, although normally
expressed in mature human smooth muscle cells, is lost
during the dedifferentiation process in culture. Arresting
cell growth by using serum-free conditions for up to
19 days also failed to induce such a current in HISM cells.
Chemicals used
Tetrodotoxin (TTX), tetraethylammonium chloride (TEA-Cl),
adenosine 5Ÿ-triphosphate (ATP, magnesium salt), guanosine
5Ÿªtriphosphate (GTP, sodium salt), guanosine-5Ÿ-O-(2-thio-
diphosphate (GDPbS, trilithium salt), cyclopiazonic acid (CPA),
creatine, Hepes, BAPTA, EGTA, NMDG-Cl, carbamylcholine
chloride (carbachol) and atropine were obtained from Sigma Chemical
Co. SK&F 96365 was obtained from Biomol Research Laboratories,
Inc. (Plymouth Meeting, PA, USA). All other chemicals were from
BDH Laboratory Supplies (AnalaR grade; Poole, Dorset, UK).
METHODS
RESULTS
Whole-cell membrane currents
Cell culture
HISM cells (ATCC no. 1692-CRL) were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) with high glucose (Gibco-BRL),
supplemented with 10% fetal calf serum (FCS), 100 u ml_1 penicillin
(Gibco-BRL) and 100 u ml_1 streptomycin. For electrophysiology,
cells were treated with 0.25% trypsin (Gibco-BRL) for 1min at 37°C,
collected by centrifugation, resuspended in fresh medium, plated
on glass coverslips and left to attach for about 10 min. Cells were used
after passages 16 and 17.
Experiments were performed on 122 human cultured
intestinal cells from passages 16–17. In 11 cells studied
using basic external PSS and K+-based, low-EGTA
pipette solution, voltage pulses applied from either _50
or _100 mV evoked time-independent currents the size
of which was a linear function of the test potential
between _90 and about 0 mV. These presumably ‘leak’
currents indicated an input resistance of about 3 GΩ. At
more positive potentials an outward current was
activated and reached a peak in about 20–100 ms in the
voltage range 0–90 mV, respectively (Fig. 1). The current
was reversibly inhibited by TEA+ with an IC50 of
Electrical recordings and data analysis
Whole-cell membrane current was recorded at room temperature
using low-resistance borosilicate patch pipettes (1–3 MΩ) and an
Axopatch 200A (Axon Instruments Inc.) voltage-clamp amplifier.
Voltage-clamp pulses were generated and data were captured using a
Digidata 1200 interfaced to a computer running the pCLAMP 6
program (Axon Instruments Inc.). Series resistance was compensated
(usually by about 80%) to produce the fastest transient without
oscillations. For sodium current (INa) recordings the signal was
filtered (4ªpole low-pass Bessel filter) at 5 kHz and sampled at
40 kHz; for cationic current these settings were 1 and 5 kHz,
respectively. No leak correction was used in either case. Data were
analysed and plotted using MicroCal Origin version 5.0 software
(MicroCal Software, Inc., Northampton, MA, USA). Values are given
9.0 0.1m
M
(n = 3) and it was also abolished when K+
was replaced by Cs+ in the pipette solution. Thus, the
current could be tentatively identified as a delayed
rectifier K+ current. No spontaneous transient outward
current (STOC)-like activity (Bolton et al. 1999),
characteristic of smooth muscle cells of various origins,
was seen in HISM cells. Moreover, caffeine (10 m
carbachol (50 µ ) application even at depolarized or
positive potentials (e.g. +10 mV) did not activate any
M) or
M
as means
S.E.M.

J. Physiol 528.3
523
Ion currents in cultured human smooth muscle cells
outward currents. Since ryanodine- and carbachol- _5315 pA at +10 mV from a holding potential of
sensitive intracellular Ca2+ stores are functional in these _100 mV) with no obvious relation to cell size. The mean
cells (Bielefeldt et al. 1997), these results suggest the value was 2.3 0.2 nA (n = 24), which corresponded to a
absence or a very low expression level of Ca2+-activated current density of 20 2 pA pF_1. The cell membrane
K+ channels. Furthermore, iberiotoxin (IbTX), a potent capacitance was on average 127 8 pF (n = 67).
and selective inhibitor of large conductance Ca2+-
Replacing external Na+ with Cs+ completely abolished
activated K+ (BKCa) channels, had no effect on IK (Fig. 1,
bottom left). In four cells IK amplitude at +100 mV in the
the inward current even though either 10 m
Ca2+ or
M
1 0 m
Ba2+ was present in the external solution (Fig. 2A
M
presence of 300 n
M
IbTX was 94 10% of control
and B). The current was also abolished by replacing Na+
(P < 0.05).
with K+ or NMDG+ (not shown).
The outward current evoked from a holding potential of
_50 mV was not affected by shifting the holding
potential to _100 mV. However, an initial fast transient
inward current appeared. To isolate and characterize this
current in the experiments described below, K+ in the
pipette and external solutions was replaced by Cs+ and
Na+, respectively.
There are two major classes of voltage-dependent Na+
channel, TTX-sensitive and TTX-resistant channels.
TTX was added to the external solution in a cumulative
manner at concentrations ranging from 0.1
while INa was measured at 10 s intervals (Fig. 2C). TTX at
1 n slightly reduced the current; at 1µthe current was
inhibited almost completely (superimposed current traces
in Fig. 2D). The IC50 value was 56 n (Fig. 2E; mean
96 21n , n = 3).
Mnto 1µ
M
M
M
TTX-sensitive Na+ current
M
M
An inward current with rapid activation/inactivation
kinetics was seen in all cells studied. However, its
maximal amplitude greatly varied (from _670 to
Superimposed TTX-sensitive currents obtained by
subtracting current traces in the presence of 1TµTX
M
Figure 1. Whole-cell membrane currents in HISM cells under quasiphysiological conditions
Families of superimposed whole-cell currents in a single cultured HISM cell evoked by 150 ms voltage
pulses applied at 10 s intervals from a holding potential of _100 mV to test potentials between _40 and
+100 mV with a 10 mV increment in control and in the presence of 10 mM TEA+ or 300 nM IbTX. I–V
relationships were constructed by measuring the peak amplitude of the outward current at each test
potential. High-K+ pipette solution and normal PSS in the bath were used.

J. Physiol 528.3
524
A. V. Zholos, C. J. Fenech, S. A. Prestwich and T. B. Bolton
from those in control are shown in Fig. 3A. Voltage pulses to +30 mV. The decay time constant decreased from
were applied from _100 mV to test potentials ranging about 10 to 2.5 ms at potentials from _10 to +30 mV,
from _70 to +90 mV in 5 mV increments. Measurable respectively. At more negative potentials where the
inward current was activated at about _30 mV, reached current was too small for reliable fitting or could not be
a peak value at +10 mV and reversed at +68 mV, very measured directly, a conventional double-pulse protocol
close to the ENa of +65 mV ([Na+]i was 10 m
; Fig. 3B). revealed an inactivation process with time constants in
M
The activation and inactivation kinetics accelerated with the range 25–14 ms at potentials from _50 to _20 mV,
membrane depolarization. Thus, the time to peak respectively (not shown). Overall the time constant
exponentially decreased from 12 to 2 ms in the range _30 decreased e-fold with depolarization by 37 mV.
Figure 2. Inward currents in a single cultured HISM cell
A, superimposed current traces elicited by depolarizing pulses to 0 mV from _100 mV in control (135 mM
Na+, 2.5 mM Ca2+) and after replacement of Na+ by Cs+ (125 mM) with 10 mM CaCl2 or BaCl2 added to the
external solution. B, I–V relationships for the peak inward current in control (•) and high-Cs+ external
solution with 10 mM Ca2+ (0) or 10 mM Ba2+ (9) in the same cell. C, peak inward Na+ current was measured
at +10 mV by applying voltage steps from _100 mV every 10 s upon cumulative application of ascending
concentrations of TTX as shown by the horizontal lines on the logarithmic concentration scale. External
solutions contained Na+ and Ca2+ but not Mg2+. D, examples of INa from the same experiment. The
concentrations of TTX applied are indicated. E, concentration–effect curve for the experiment illustrated
in C and D. Data points represent relative current (ITTX/IControl) fitted by the logistic function with an IC50
value of 56 nM. ITTX is the current in TTX.

J. Physiol 528.3
525
Ion currents in cultured human smooth muscle cells
Peak sodium conductance was estimated by dividing the Boltzmann distribution), the V1/2 and k values could be
peak current amplitude by the driving force (E _ ENa, estimated to be _13.1 and _9.4 mV, respectively, for the
where E is the membrane potential) at each test potential example shown in Fig. 3C.
(Fig. 3C). The half-activation potential (V1/2) was found to
The steady-state inactivation of the current was assessed
by holding the membrane potential at different values
between _120 and _15 mV for 10 s followed by a voltage
step to +10 mV. Superimposed current traces obtained
be _3.2 1.4 mV and the slope factor (k) was
_6.7 1.0 mV (n = 19). The activation kinetics of the
Na+ current was not analysed, but assuming an m3
process (e.g. G/Gmax = m3, where Gmax is the maximal
with this protocol are shown in Fig. 3D. Plotting the peak
conductance and the activation parameter m obeys the
Figure 3. Voltage-dependent properties of TTX-sensitive inward Na+ current in external solution
with Ca2+ but no Mg2+
A, superimposed current traces obtained by paired subtraction of currents afteMr T1TµX application
from those in control. The holding potential was _100 mV; 30 ms voltage steps were applied to test
potentials ranging from _70 to +5 mV (left) and from +10 to +90 mV (right) in 5 mV increments. B, I–V
relationship for the peak INa shown in A. C, activation curve obtained by dividing the peak current
amplitude by the driving force at each test potential (E _ ENa, where ENa = +65 mV) and fitted by a
Boltzmann function with the following best fit parameters: maximal conductance, Gmax = 34 nS;
potential of half-maximal activation, V1/2 = 0 mV; slope factor, k = _7.3 mV. D, superimposed current
traces elicited by 25 ms voltage steps to +10 mV applied from various holding potentials (_120 to
_15 mV in 5 mV increments). E, steady-state inactivation of INa for the experiment illustrated in D. Data
points were fitted by a Boltzmann function with a potential of half-maximal inactivation of _57.4 mV
and a slope factor of 9.8 mV.

J. Physiol 528.3
526
A. V. Zholos, C. J. Fenech, S. A. Prestwich and T. B. Bolton
current as a function of the preceding holding potential and _180 mV as in Fig. 5A and B or a slow voltage ramp
(e.g. Fig. 3E) revealed a half-inactivation potential of in the same range as shown schematically in Fig. 6A
_61.9 2.2 mV and a slope factor of 9.5 0.6 mV (bottom). The first current was inwardly rectifying with
(n = 1 0).
instantaneous activation and deactivation kinetics and
no obvious inactivation except at very positive potentials
(Fig. 5A and B). Voltage steps and slow voltage ramps
produced very similar I–V relationships (Fig. 5C, circles
and continuous lines, respectively). We termed this
hyperpolarization-activated current IHA. A second
current was seen upon a voltage step to _40 mV from a
more negative test potential as in Fig. 5B or following the
voltage ramp as in Fig. 6A. We termed this
depolarization-activated current IDA. It showed rapid
inactivation kinetics and thus might have been
interpreted as a tail current due to the deactivation of the
inwardly rectifying conductance, but further
experiments showed that this was not the explanation.
Both currents progressively increased in size with time
after divalent cation removal (Fig. 6B, top panel).
However, the time course of their increases was different.
Recovery from inactivation was measured by applying a
70 ms depolarizing pulse to +10 mV followed by a short
test pulse to +10 mV with a variable delay (Fig. 4A,
bottom trace). The recovery process could be well
described by a single exponential function (dashed line in
Fig. 4A; see also Fig. 4B). There was an e-fold change in
the time constant per 15.7 mV in the example shown in
Fig. 4B and C. On average the time constant describing
the recovery process increased e-fold per 17.3 4.2 mV
(n = 4).
Cationic currents
When the external solution was changed to a divalent
cation-free, 130 m
Na+ solution two additional currents
M
were observed. The voltage protocol used was either a
series of steps from _40 mV to potentials between +100
Figure 4. Recovery of INa from inactivation
A, INa elicited by a 70 ms prepulse and a 10 ms test pulse to +10 mV applied from _100 mV with a
variable interpulse interval, t (as shown below). Capacitance transients have been removed for clarity.
Peak INa during the recovery process could be well fitted by a single exponential function with a time
constant of 12.3 ms as shown by the dashed line. B, relative INa plotted against interpulse interval on a
semilogarithmic scale at different membrane potentials (from left to right, _120 to _60 mV in 10 mV
increments) and fitted by single exponential functions as shown by the continuous lines. C, voltage
dependence of the time constant characterizing INa recovery from inactivation in a single experiment,
fitted by a single exponential function with an e-fold increase in r per 15.7 mV.

J. Physiol 528.3
527
Ion currents in cultured human smooth muscle cells
The inwardly rectifying current reached a maximum 75 s IC50 value in the same cell was 20 µ
M
and the
after external divalent cation removal (e.g. Fig. 6B, top concentration–effect curve was less steep (Fig. 6B,
panel, circles) whereas in the same cell the current seen bottom). These observations indicated that the currents
upon the voltage step to _40 mV continued to increase in were not related. Overall statistics for the occurrence of
size and stabilized in different cells after about 100–200 s these currents in different cells also supported this
(e.g. Fig. 6B, top panel, triangles). Furthermore, the conclusion. Thus, in experiments with Na+ or K+ as the
dependence of IHA and IDA on the external calcium main cation in the external solution, in only 35% of cells
concentration was different. The currents stabilized 50 s studied (14 out of 40) were the two currents observed
after Ca2+ readmission to the external solution. IDA was simultaneously. IHA was present in the majority of these
inhibited with an IC50 of 311 n whereas for IHA the cells whereas IDA was seen less frequently (in 35 and 16 out
M
Figure 5. Inward currents in cultured HISM cells developing in divalent cation-free external
solution
A and B, superimposed current traces elicited by 800 ms duration voltage steps from _40 mV to test
potentials between +100 and _180 mV in 10 mV increments in control (130 mM Na+, 2.5 mM Ca2+, 1.2 mM
Mg2+; A) and after external Ca2+ and Mg2+ removal (B, denoted as IHA). Note the instantaneous activation
and deactivation of IHA as well as the noisy appearance of the current at negative potentials. The current
seen upon repolarization to _40 mV is denoted as IDA. C, I–V relationships for currents measured at the
end of the pulse (1 and 0) and, in the same cell, by applying 1s duration voltage ramps from +100 to
_180 mV (continuous lines). The ramp protocol is illustrated in Fig. 6A.

J. Physiol 528.3
528
A. V. Zholos, C. J. Fenech, S. A. Prestwich and T. B. Bolton
order: K+ > Cs+ > Na+ for IHA (Fig. 6C) and
Na+ > K+ >> C s + for IDA (Fig. 6D). Similar results were
obtained from five cells. Substitution with the large
impermeable NMDG+ cation completely abolished both
IHA (Fig. 6C; note that control current was recorded in
of 40 cells studied, respectively). In 20 cells studied using
Cs+ as the main external cation, IDA was undetectable
whereas IHA was seen in 19 cells. These observations
implied different ion selectivity of the channels
underlying these currents. To investigate this further, ion
substitution experiments were performed. In the
Na+ solution containing 2.5 m Ca2+) and IDA (Fig. 6D;
M
experiments illustrated in Fig. 6C and D, 130 m
Na+ in
M
note the small inward current in control solution, Na+
Ca2+, compared to the NMDG+-based
the external solution was replaced by equimolar amounts
of Cs+, K+ or NMDG+. The sizes of the current in the
different solutions were in the following descending
with 2.5 m
M
solution). Moreover, the reversal potential was close to
0 mV and remained almost the same: +5.5 mV with Na+,
Figure 6. Kinetics, [Ca2+]o dependence and ion permeation properties of IHA and IDA
A, typical currents during a slow voltage ramp from +100 to _180 mV and a voltage step to _40 mV
slowly developing after Ca2+ and Mg2+ removal from the external solution. Superimposed current traces
were recorded at 25 s intervals. B, top, maximal amplitudes of inward currents at _180 mV (0, IHA) and
at _40 mV after the ramp (8, IDA) plotted against time where time zero represents the moment of Ca2+ and
Mg2+ removal from the external solution. Bottom, dependence of IHA (0) and IDA (8) in the same cell on the
external free Ca2+ concentration. Relative currents were fitted by logistic functions with IC50 values of
20 µM for IHA and 311 nM for IDA. Cand D, external cation substitution experiments were performed on two
different cells after IHA (C) and IDA (D) had been stabilized in a divalent cation-free external solution. The
control I–V relationship for IHA (voltage protocol as in A) and control current response for IDA (voltage step
from _100 to _40 mV) were obtained in high-Na+, 2.5 mM CaCl2-containing external solution. Traces
labelled ‘Na+’ were obtained after Ca2+ removal whereas all other traces were obtained in solutions with
Na+ replacement as described in the Methods. High-Cs+ pipette solution was used.

J. Physiol 528.3
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Ion currents in cultured human smooth muscle cells
+3.3 mV with Cs+ and +2.6 mV with K+ in the external
solution. In three cells expressing IHA alone, changing the
pH of the external solution from 7.4 to 6.4 or 8.4 had
almost no effect on the amplitude of the current or its
reversal potential (data not shown).
(Almers et al. 1984), chick embryo ectoderm (Li et al.
1994), cardiac muscle (Mubagwa et al. 1997) and Xenopus
oocytes (Zhang et al. 1998).
Another possibility was that in Ca2+-free external
solution the intracellular stores could be passively
depleted and this could account for the slow development
of IHA and IDA due to the opening of calcium release-
activated channels (CRACs). Thus, we performed
experiments to see whether Ca2+ store depletion could
induce such currents per se.
The above results clearly indicate that IHA and IDA are
carried via distinct cation channels which differ in their
relative permeabilities to Na+, K+ and Cs+ and in their
voltage and extracellular Ca2+ dependence.
Pharmacological properties of IHA and IDA cationic
currents
In these cultured intestinal smooth muscle cells both
ryanodine- and InsP3-sensitive Ca2+ stores have been
identified and the cholinergic agonist carbachol has been
shown to release Ca2+ from both pools (Oh et al. 1997;
Bielefeldt et al. 1997). Thus, we tested further the effects
of carbachol. In the presence of Ca2+, carbachol
application induced no or a very small inward current. It
is also interesting to note here that the delayed rectifier
K+ current was strongly inhibited by carbachol (by 60%
It is now well established that a variety of cationic
channels are commonly expressed in various smooth
muscles. The gating events which open these channels are
also diverse and include membrane stretch, Ca2+ release
and/or a rise in [Ca2+]i, receptor and Gªprotein activation,
a change in membrane polarization, protein kinase C
activation, etc. Our observation that IHA and IDA were
activated by Ca2+ removal from the external solution
indicated a crucial role of external Ca2+ in the channel
gating. A wide variety of cell types respond to external
Ca2+ removal by generating cationic currents or non-
selective currents carried by both cations and anions. The
known examples include epithelium (Van Driessche &
Zeiske, 1985; Desmedt et al. 1993), skeletal muscle
at 50 µ ); the inhibition was voltage independent
M
between 0 and +80 mV. Such an effect has been
previously demonstrated in freshly isolated gastric
myocytes (Lammel et al. 1991) as well as for the cloned
smooth muscle-derived delayed rectifier potassium
channels (Vogalis et al. 1995).
Figure 7. Effects of carbachol on IHA and IDA
A, the control I–V relationship was measured as described for Fig. 6C. After IHA had developed in divalent
cation-free, high-Cs+ external solution, it was rapidly potentiated by carbachol (CCh) application. IDA was
abolished in Cs+-containing external solution and was inactivated at _40 mV. B, in a different cell, IDA
measured at _40 mV in divalent cation-free, high-Na+ external solution was strongly inhibited by
carbachol.

J. Physiol 528.3
530
A. V. Zholos, C. J. Fenech, S. A. Prestwich and T. B. Bolton
Figure 8. Pharmacological properties of IHA and IDA
A, 5 min treatment with 30 µM CPA failed to induce an inward IHA (left) or IDA (right) in 2.5 mM Ca2+, high-
Na+ external solution (n = 5), but both currents were readily activated following Ca2+ removal. Note that
at positive potentials current could be activated by CPA. B, SK&F 96365 at concentrations of up to
100 µM did not inhibit inward IHA (left), but completely abolished IDA (right) in the same cell (n = 7).
Current at positive potentials at which it could be activated by CPA (left panel in A) was also inhibited
by SK&F 96365. C and D, IDA (right) was more sensitive to the inhibitory action of Gd3+ or La3+ compared
to IHA (left) but both currents were abolished by 100 µM of either blocker. In all panels IHA and IDA were
measured in the same cells.

J. Physiol 528.3
531
Ion currents in cultured human smooth muscle cells
However, after IHA had developed in Ca2+-free solution, it Ca2+ stores. In four cells tested, application of CPA at
was rapidly increased by carbachol application in a dose- 30 µ for 5–8 min failed to induce inward cationic
dependent manner; carbachol at 1
µ was nearly currents when Ca2+ was present in the external solution
M
M
maximally effective (Fig. 7A). The amplitude of IHA in the but both currents could be readily induced in the same
presence of saturating agonist concentrations (10– cells in Ca2+-free solution (Fig. 8A). Thus, it appeared that
100 µ
2.77 0.50 (n = 5). The effect of carbachol was mechanism, was insufficient for IHA or IDA activation as
practically abolished in the presence of 1µatropine the channels are primarily controlled (suppressed) by
(ratio of amplitudes, 1.09 0.05 (n = 4) with and without external calcium ions. It was also notable in all cells tested
atropine plus 10 µ carbachol) or in cells where GDPbS that CPA did induce an outward current. Its amplitude at
(2 m
M
) relative to that before carbachol application was Ca2+ store depletion alone, if this was an underlying
M
M
M
) was allowed to diffuse into the cell from the very positive potentials was the same as that of IHA
pipette solution for about 5 min to inhibit Gªproteins during subsequent Ca2+-free solution application
prior to carbachol application (there was little increase in (Fig. 8A).
IHA; the ratio of amplitudes in these cells was 1.06 0.08
SK&F 96365, a blocker of store-operated Ca2+ channels
(n = 4) with 10 µ carbachol). Thus, both effects were
M
(Clementi & Meldolesi, 1996), suppressed IDA with a half-
maximal inhibitory concentration of 9 µ but was
ineffective in blocking inward IHA at concentrations up to
100 µ (Fig. 8B). Interestingly, an outward current at the
same potentials at which it could be induced by CPA was
statistically significant (P < 0.03; two-tailed unpaired
t test) indicating the involvement of muscarinic
receptors/Gªproteins in the signal transduction path-
ways. As mentioned above, IDA occurred very
infrequently but in two cells where this current was
M
M
inhibited by SK&F 96365. Also, at 100 µ SK&F 96365
M
present the same carbachol concentrations (10–100 µ )
M
almost completely blocked the TTX-sensitive Na+
current; the inhibition was reversible (ron = 7.5 s;
roff = 103 s).
strongly inhibited and finally abolished IDA (one example
is shown in Fig. 7B).
Cyclopiazonic acid (CPA), an inhibitor of the sarco-
(endo)plasmic reticulum Ca2+ pump (SERCA), is widely
used as a pharmacological tool to deplete intracellular
Gadolinium and lanthanum are useful pharmacological
tools as antagonists of mechano-sensitive (e.g. stretch- or
Figure 9. Amplitude and kinetic relationships between INa and IDA
Current traces were recorded in a high-Na+, divalent cation-free solution upon voltage steps from
_100 mV to various test potentials indicated beside each trace. IHA was 160, 122 and 81 pA at _100, _70
and _35 mV, respectively.

J. Physiol 528.3
532
A. V. Zholos, C. J. Fenech, S. A. Prestwich and T. B. Bolton
swelling-activated) cationic channels (Hamill & McBride, Since IDA showed steady-state inactivation properties
1996), as well as external Ca2+-blockable cationic channels very similar to those for INa in these cells (see Fig. 10C) we
(Van Driessche et al. 1988; Mubagwa et al. 1997; Zhang et also considered the possibility that modulation of Na+
al. 1998). In 13 HISM cells tested, IDA was found to be channel gating in Ca2+-free solution could somehow be
more sensitive to the blocking action of Gd3+ and La3+ involved (Armstrong, 1999; Armstrong & Cota, 1999).
compared to IHA, the latter being completely inhibited However, TTX at 1µ had no effect on IDA. This current
M
only at 100 µ
M
(Fig. 8D).
was also distinct from INa in its kinetics and voltage
dependence of activation. Figure 9 shows current traces
Voltage-dependent properties of IDA
Figure 10. Comparison of the voltage dependence of activation and inactivation of INa and IDA
A and B, families of superimposed current traces and voltage protocols used to study the voltage
dependence of activation (A) and inactivation (B). Measurements were made of INa at its peak and
activation of IDA 30 ms after stepping to various potentials from a holding potential of _100 mV; at 30 ms
INa is largely inactivated (cf. Fig. 3) but IDA is close to its peak value. In B the test potential was +10 mV,
close to the reversal potential (Vrev) for IDA, thus minimizing its contribution during measurements of INa
inactivation. Again, a 30 ms pulse allows INa to inactivate leaving IDA close to its peak value. C, open and
filled circles show, respectively, normalized INa amplitude from B (hNa) and relative Na+ conductance
calculated from INa amplitudes in A (mNa) as described for Fig. 3C, respectively. Filled and open triangles
show relative tail current amplitudes from A (mCat) and B (hCat), respectively, measured by fitting single
exponential functions and extrapolating to time zero at the end of each test pulse. Data points were fitted
by Boltzmann functions with the following best fit parameters: INa activation: V1/2 = _11.1 mV;
k = _8.3 mV; INa inactivation: V1/2 = _74.5 mV, k = 8.8 mV; IDA activation: V1/2 = _45.8 mV,
k = _5.0 mV; IDA inactivation: V1/2 = _69.2 mV, k = 8.1mV. This cell was in 130 mM Na+, divalent
cation-free solution; IHA was small (_145 and +155 pA at _100 and +90 mV, respectively).

J. Physiol 528.3
533
Ion currents in cultured human smooth muscle cells
recorded in high-Na+, divalent cation-free external
solution upon stepping from the holding potential of
_100 mV to various levels indicated beside each current
trace. Activation of IDA occurred at potentials more
negative than activation of INa (e.g. _60 mV). Since IDA
activation was much slower than that of INa these
currents produced two distinct peaks (e.g. between _45
and _35 mV). The rate of IDA inactivation was also
considerably slower. At more positive test potentials, IDA
was masked by a generally much larger INa. Thus, to
study the voltage dependence of its activation and
inactivation we employed the protocols shown
schematically in Fig. 10A and B, respectively. These
voltage-dependent parameters for IDA were assessed by
measuring tail current amplitude at _100 mV after
voltage steps to different test potentials (activation;
Fig. 10A) or after a voltage step to +10 mV applied from
different holding potentials (inactivation; Fig. 10B). The
amplitude of the tail current was measured by fitting a
single exponential function with extrapolation to the
beginning of repolarization. Normalized values are shown
in Fig. 10C by triangles, with open symbols for
inactivation and filled symbols for activation. For
comparison, steady-state activation and inactivation
curves for INa in the same cell are also shown; these were
obtained as described above (e.g. Fig. 3). Steady-state
inactivation for the two currents was surprisingly similar
both in the slope and in the position on the voltage axis
(only 5 mV difference in the V1/2 values). However, the
activation curve for IDA was steeper and positioned about
35 mV more negative than that for INa. Thus, a small
steady IDA (‘window current’) can be generated in the
voltage range _60 to _30 mV under conditions when
Ca2+ stores are depleted (compare to Fig. 9).
The position of the activation curve for IDA on the voltage
axis is very similar to that for the muscarinic cationic
conductance in guinea-pig ileal smooth muscle cells
(compare with Inoue & Isenberg, 1990; Zholos & Bolton,
1994). Given that both currents are cation currents, IDA
might be expected to show a similar U-shaped I–V
relationship. In the experiment illustrated in Fig. 11A,
IDA was measured by applying voltage steps from
Figure 11. I–V relationships for IDA
A and B, voltage protocols and superimposed current traces recorded in the same cultured HISM cell using
high-Cs+ pipette and high-K+, divalent cation-free external solution in which INa is abolished and IDA can
be measured. IHA was relatively small in this cell: measured by applying voltage ramps from _40 mV to
inactivate IDA, IHA amplitude was _427 pA at _70 mV and 315 pA at +60 mV. In B steps were in the
range _70 to +60mV. Tail current was flat at +20 mV. C, I–V relationships for the peak IDA in A (0) and
instantaneous tail current amplitude in B (1), measured as described for Fig. 10B. Tail current reversed
at +23 mV which was 3 mV more positive than the peak IDA reversal potential. This discrepancy could
arise as a result of some contamination of the peak IDA by IHA, as explained above, for which Vrev ≈ 0 mV.

J. Physiol 528.3
534
A. V. Zholos, C. J. Fenech, S. A. Prestwich and T. B. Bolton
_100 mV to various test potentials. The instantaneous up to 19 days did not abolish INa but substantially
I–V relationship for this current was also evaluated in the accelerated its inactivation kinetics; for example at
same cell by stepping to 0 mV (maximal activation) +10 mV the decay time constant decreased from 5.2 to
followed by voltage steps to between _70 and +60 mV 0.9 ms (data not shown).
(Fig. 11B). Figure 11C compares I–V relationships
The overall properties of INa in cultured HISM cells
obtained using these protocols. The instantaneous I–V
appear very similar to those in freshly isolated human
colonic myocytes except for an approximately 7-fold
higher IC50 value for TTX (Xiong et al. 1993). In both
cases the currents peaked at about 0 mV and inactivated
within about 10 ms. The availability curves had similar
slopes and V1/2 values but the activation curve in HISM
was positioned 19 mV more positive in HISM cells
relationship (1) is nearly linear implying an almost linear
dependence of single channel current amplitude on the
membrane potential (constant single channel
conductance), whereas peak IDA showed a bell-shaped
voltage dependence (0) declining almost to the zero level
at very negative potentials (compare with Fig. 5 of Inoue
& Isenberg, 1990). The reversal potential in the two cases
compared to native cells without any significant change
was nearly the same.
in the voltage dependence (slopes, 6.7 vs. 7.6 mV,
respectively). There was also a clear indication of the
overexpression of Na+ channels in cultured cells as the
DISCUSSION
current density on average was 20 times higher compared
to that of freshly isolated human cells (Xiong et al. 1993).
Smooth muscle cell cultures offer a valuable approach to
study the mechanisms and potential regulatory
pathways controlling cell differentiation (Owens, 1995).
However, substantial modification of cell properties
means that careful studies are required in each particular
case and the results obtained may not represent in vivo
properties of fully differentiated cells. In the present
patch-clamp study we investigated the electro-
physiological behaviour of cultured HISM cells, which
until now were poorly characterized.
The lack of voltage-gated Ca2+ channels (Fig. 2A and B)
in these cells is clearly the result of altered properties of
cultured HISM cells as compared with their native
counterparts. The L-type channel proteins are
ubiquitously expressed in all freshly isolated gastro-
intestinal smooth muscle cells studied, including human
jejunal and colonic myocytes (Farrugia et al. 1995; Xiong
et al. 1995). It must be noted that in a previous study the
inward current in cultured HISM cells was erroneously
identified as a voltage-gated Ca2+ current via L-type Ca2+
channels despite its atypical rapid inactivation for such a
current. No ion substitution tests were performed and
this conclusion was drawn from the partial inhibition by
BKCa channels are ubiquitously expressed in visceral
smooth muscles giving rise to STOC discharge or large
outward currents in response to intracellular Ca2+ release
(Bolton et al. 1999). In cultured HISM cells both
ryanodine- and InsP3-sensitive Ca2+ stores are functional
(Oh et al. 1997), but under conditions of low intracellular
Ca2+ buffering we observed neither STOCs nor outward
currents in response to caffeine or carbachol. This
suggests that BKCa channels are not expressed in these
cells, which is further confirmed by the lack of block by
IbTX (Fig. 1). However, it is unlikely that these channels
are lost in culture since in cells freshly dispersed from
human jejunum, Ca2+-dependent K+ channels are also
lacking. The major difference was in the activation range
for the TEA+-sensitive K+ current, which was about
60 mV more positive in cultured cells compared to freshly
isolated jejunal cells (Farrugia et al. 1993).
Upon carbachol application, even at high concentrations,
muscarinic receptor cationic current, ICAT, with a
characteristic U-shaped voltage dependence was not
observed in HISM cells. Since in guinea-pig ileal cells ICAT
is strongly inhibited by external divalent cations (Zholos
& Bolton, 1995), Ca2+ and Mg2+ were removed in an
attempt to unmask this current. Though ICAT was not
revealed under these conditions we found two other
cationic currents previously not seen in freshly
dissociated smooth muscle cells. Under divalent cation-
free conditions they developed very slowly and initially
almost in parallel (Fig. 6A and B). At later times the
current termed IHA stabilized whereas the other current,
termed IDA, continued to increase. This was the first
indication that different ion channels mediate these two
currents. IDA turned out to be much more sensitive to
[Ca2+]o (Fig. 6B). The channels also had different ion-
selectivity profiles and different voltage-dependent and
pharmacological properties.
nifedipine (20 µ ) and verapamil (Bielefeldt et al. 1996).
M
In our experiments this fast inward current persisted in a
Ca2+-free external solution (e.g. Figs 9 and 10) and was
abolished in Na+-free solutions (replacement by Cs+, K+
or NMDG+; e.g. Figs 2A and 11A). Moreover, the current
was TTX sensitive (IC50 ≈ 1 00 n ). Many different
M
smooth muscle cells normally express both Ca2+ channels
and voltage-gated, TTX-sensitive Na+ channels (e.g. rat
ileum, Smirnov et al. 1992; for review see Kuriyama et al.
1998). Na+ channels may also be expressed in cultured
smooth muscle cells, usually in a relatively minor
population of cells, even though they are not observed in
their native counterparts (e.g. Snetkov et al. 1996). TTX-
sensitive Na+ channels were present in all HISM cells
tested though INa greatly varied in amplitude with no
obvious relation to cell size. It is interesting to note that
arresting cell growth by using serum-free conditions for
The slow appearance of the currents in divalent cation-

J. Physiol 528.3
535
Ion currents in cultured human smooth muscle cells
free medium could indicate the involvement of passive affect the cationic currents. The voltage-dependent
Ca2+ store depletion in their generation. Thus, the availability curves for INa and IDA were strikingly similar
channels could be potentially related to CRACs through (Fig. 10C), but other properties were different and it is an
which capacitative refilling of Ca2+ stores is believed to important observation that all three currents could be
occur. This has been found to be a dominant Ca2+ entry observed in the same cell. Moreover, INa was abolished in
pathway in a large variety of cells, particularly in non- K+ solutions but both cationic currents remained.
excitable cells (reviewed by Parekh & Penner, 1997).
The other possibility is that Ca2+ removal unmasks
Store-operated Ca2+ influx has also been reported in
cationic channels (e.g. Van Driessche & Zeiske, 1985;
cultured smooth muscle cells such as A7r5 vascular cells
Mubagwa et al. 1997). Activation of hemi-gap-junctional
channels has recently been implicated in Xenopus oocytes
(Blatter, 1995; Byron & Taylor, 1995) and the DDT1MF-
2 cell line (Ufret-Vincenty et al. 1995).
(Zhang et al. 1998), but these channels are permeable to
ICRAC is difficult to measure directly in smooth muscle anions and even to large organic monovalent cations such
cells. However, recent studies on other cell types have as NMDG+, which is clearly not the case in HISM cells
established that monovalent cation outward currents can (e.g. Fig. 6C and D). Also, the voltage dependence and
be generated by CRACs even when Ca2+ is present in the channel kinetic properties of HISM cells and oocytes are
external solution (Hoth, 1996). When the external free different.
Ca2+ concentration was reduced to micromolar levels in
In ventricular myocytes, Mubagwa et al. (1997) described
the absence of Mg2+, ICRAC in Jurkat T-cells was
a novel Ca2+-blockable cationic current with properties
significantly increased (Lepple-Wienhues & Cahalan,
remarkably similar to those of IHA in HISM cells. The
1996) and single CRAC conductance for Na+ was much
current was blocked by Ca2+ with an IC50 of 60 µ
M
higher compared to the Ca2+ current through CRACs
(Kerschbaum & Cahalan, 1999). Using CPA, a sarco-
endoplasmic reticulum Ca2+ pump (SERCA) inhibitor, to
deplete the store we found that an outward current
developed and the same component of the current was
inhibited by SK&F 96365, a blocker of store-operated
Ca2+ channels (Fig. 8A and B). However, beyond this
there were more differences than similarities between IHA
and IDA in HISM cells and ICRAC in other cells. To
summarize, it appears that some properties of a CRAC
current are shared with IHA (e.g. voltage dependence,
kinetics, inward rectification, the lack of inactivation in
Ca2+-free solution, similar IC50 values for inhibition by
external Ca2+, effects of carbachol) whereas other
properties are shared with IDA (e.g. the blocking action of
Gd3+, La3+ and SK&F 96365, and the sequence of
conductance Na+ > K+ >> C s + is identical to that in
Jurkat T cells). Thus, an amplified CRAC component may
be present in divalent cation-free solution but the
properties of neither IHA nor IDA are the same as those
described for ICRAC in other cell types.
(compare to 20 µ for IHA). Thus, the authors postulated
M
the existence of a high-affinity binding site for Ca2+ at or
near the extracellular site of the cationic channel, so that
the slow off-rate could explain the slow development
(> 5 min) of the current, which is also the case for IHA. IDA
developed even more slowly, consistent with an even
lower apparent dissociation constant for Ca2+ of about
0.3 µ
inward rectification, and have a very similar noisy
appearance, amplitude and instantaneous activation
M. IHA and cationic current in cardiac cells both show
/
deactivation during voltage jumps (compare our Fig. 5B
with Fig. 2A in Mubagwa et al. 1997). Both currents could
be carried by monovalent cations in the same sequence
K+ > Cs+ > Na+ >> NMDG+, but not by Cl_, and were
abolished in the presence of 100 µ
Gd3+.
M
The physiological relevance of such a conductance in cells
in vivo exposed to [Ca2+]o in the millimolar range remains
largely unclear. However, in cardiac cells there is a
difference in the [Ca2+]o sensitivity between intact tissue
and single cells. Also, Mubagwa et al. (1997) raised the
possibility that these channels can be physiologically
regulated by agonists (though they did not observe any
response to b-adrenergic or muscarinic receptor
stimulation) or change their calcium sensitivity under
pathophysiological conditions such as ischaemia or ‘Ca2+
paradox’. Our findings that carbachol application can
strongly potentiate IHA via a muscarinic receptor/
Gªprotein pathway support this hypothesis. Our first
demonstration of IHA in smooth muscle cells also shows
that such a current is not tissue specific. Moreover, IDA in
HISM cells is a novel Ca2+-blockable cationic current with
Since no stimulation of the cells was employed
(e.g. mechanical stimuli or agonists) to evoke these
currents, the remaining possibility is that IHA and IDA
belong to the family of Ca2+-blockable cationic currents
which appear to be ubiquitous in various cell types but so
far have not been described in smooth muscle cells. The
opening of such channels is believed to produce the
membrane depolarization commonly occurring in Ca2+-
free solutions and two mechanisms have been proposed to
explain this phenomenon.
One possibility is that the removal of external Ca2+ alters no resemblance to previously described currents. Thus,
the selectivity and/or gating properties of Ca2+ or Na+ even in the same cell distinct channels can mediate
channels (e.g. Almers et al. 1984; Armstrong, 1999; cationic fluxes in divalent cation-free solutions but their
Armstrong & Cota, 1999). In HISM cells this seems physiological function remains to be defined. HISM cells
unlikely since Ca2+ channels are absent and TTX did not may provide a useful experimental model for future

J. Physiol 528.3
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A. V. Zholos, C. J. Fenech, S. A. Prestwich and T. B. Bolton
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Acknowledgements
This work was supported by grant number 051162/Z from The
Wellcome Trust.
Corresponding author
T. B. Bolton: Department of Pharmacology and Clinical
Pharmacology, St George’s Hospital Medical School, London
SW17 0RE, UK.
Email: t.bolton@sghms.ac.uk

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