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Department of Physiology, University of Munich, D-80336 Munich, Germany
1Correspondence: Department of Physiology, University of Munich, Pettenkoferstr. 12, D-80336 Munich, Germany. E-mail: c.alzheimer{at}lrz.uni-muenchen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ATP-evoked change of membrane potential • P2Y2 receptor and intracellular Ca2+ release • Ca2+-activated Cl- channels • hSK4 • proliferation and differentiation of keratinocytes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
.
Whereas the biophysical and pharmacological properties of these
channels have been studied in some detail, much less is known about
their functional significance. A recent study demonstrated that
K+ channel activity is associated with
Ca2+-induced differentiation of keratinocytes
(6)
, suggesting that activation or inhibition of
individual ion currents and the concomitant change in membrane
potential might influence the growth and differentiation properties of
keratinocytes. This notion is supported by studies with other types of
nonexcitable cells in which K+ channels were
implicated in cell proliferation (7
8
9)
. As a first step
to establish a link between electrophysiological and molecular events,
we investigated whether extracellular mediators acting on keratinocytes
under different pathological conditions are capable of altering their
membrane potential in a significant fashion. We examined the effects of
ATP, bradykinin, and histamine, since keratinocytes are typically
exposed to these substances after skin injury and during inflammatory
skin disease and allergic reactions, respectively. To measure the
membrane potential without perturbing the intracellular milieu of the
keratinocytes under study, we used the perforated patch variation of
the whole-cell recording configuration. Experiments were performed on
HaCaT cells, an immortalized, nontumorigenic human keratinocyte cell
line that maintains partial differentiation capacity in
vitro and full differentiation capacity in vivo
(10)
. Our data indicate that ATP, bradykinin, and
histamine cause a dramatic and long-lasting hyperpolarization of the
membrane potential of HaCaT keratinocytes, preceded by a smaller and
transient depolarization. The pharmacological profile of this biphasic
voltage change suggested the involvement of
Ca2+-activated Cl- and
K+ channels. Reverse transcription-polymerase
chain reaction (RT-PCR) showed that HaCaT keratinocytes express the
Ca2+-activated Cl-
channels CaCC2 and CaCC3(CLCA2) as well as the
Ca2+-activated K+ channel
hSK4. Supporting the notion that K+
channel-mediated changes in membrane potential bear significance on the
cellular biology of keratinocytes, we found that hSK4 mRNA expression
is strongly down-regulated in confluent cells that have undergone
partial differentiation. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Electrophysiology
For electrophysiological recordings, culture medium was replaced
with standard bath solution (SBS) and the dishes were placed on the
stage of an inverted microscope (Axiovert, Zeiss). SBS contained (in
mM): 130 NaCl, 3 KCl, 2 CaCl2, 2
MgCl2, 25 HEPES/NaHEPES, and 10 D-glucose, pH
7.4. All recordings were made at room temperature (21–24°C). Patch
pipettes were fabricated from borosilicate glass using a two-stage pull
protocol on a horizontal puller (DMZ, Zeitz, Germany) and filled with a
solution containing (in mM): 135 K gluconate, 10 KCl, 1.6
Na2HPO4, 0.4
NaH2PO4, 0.73
CaCl2, 1.03 MgCl2, 1 EGTA,
14 HEPES/NaHEPES, and 100 mg/l nystatin, pH 7.2.
Ca2+ activity was 10-7 M
in this solution. Nystatin was freshly prepared every day from stock
[50 mg/ml in dimethyl sulfoxide (DMSO)]. We did not omit nystatin
from the tip of the pipette, since in our hands nystatin did not impair
seal formation. In one set of experiments, gramicidin (50 mg/l, freshly
prepared from a stock solution of 10 mg/ml in DMSO) was used instead of
nystatin. Patch pipettes had a resistance of 5–8 M
in the bath.
After formation of a seal in the G range (typically 1.5–2 G),
the amplifier was switched to current clamp mode and membrane
potentials attained stable values within 3–5 min. Electrophysiological
signals were recorded, amplified, and digitized with the use of an
Axopatch 200 amplifier in conjunction with a TL-1 Labmaster interface
and AXOTAPE software (Axon Instruments, Foster City, Calif.). Data are
expressed as mean ± SE. Statistical analysis (one-way
ANOVA with Bonferroni post-test comparison) was done with the use of
Graphpad prism 2.0.
Flash photolysis of caged IP3
In experiments in which intracellular Ca2+
was elevated by flash photolysis of caged IP3,
recordings were performed in the whole-cell configuration using a
pipette solution of the following composition (in mM): 120 K gluconate,
20 KCl, 1 MgCl2, 10 HEPES/NaHEPES, 2
Na2ATP, 0.3 NaGTP, (pH 7.2), and 10–100 µM
caged IP3. After 
5–7 min of whole-cell
recording, caged IP3 was released using a flash
of UV light generated by means of a xenon arc lamp (75 W, excitation
filter 330±40 nm), which was connected to the epifluorescence port of
the microscope. The UV light beam directed through a 40x phase
contrast objective was focused on the keratinocyte under study.
Duration of the flash (1 s) was controlled with the use of a Uniblitz
shutter (Vincent Assoc., Rochester, N.Y.).
Test solutions and chemicals
In experiments in which the entire bath solution was exchanged,
a conical insert made of plexiglas containing multiple inlet lines and
an outlet line was lowered into the dish to reduce the volume of the
recording chamber to 1 ml. Inflow was gravity dependent and controlled
by magnetic valves; outflow was effected by negative pressure provided
by a vacuum pump. In addition to SBS, the following bath solutions were
used (in mM): Na+-free bath solution, 120 HCl,
145 N-methyl D-glucamine (NMDG), 3 KCl, 2 MgCl2,
2 CaCl2, 25 HEPES/NaHEPES, 10 D-glucose, pH 7.4;
low Cl- solution, 130 Na-gluconate, 3 KCl, 2
MgCl2, 2 CaCl2, 25
HEPES/NaHEPES, 10 D-glucose, pH 7.4;
Ni2+-containing solution, 125 NaCl, 5
NiCl2, 3 KCl, 2 MgCl2, 2
CaCl2, 25 HEPES/NaHEPES, 10 D-glucose, pH 7.4.
All other substances were applied under visual control directly onto
the keratinocytes under study by means of a remote controlled,
solenoid-operated Y-tube system (diameter of outlet tube 100 µm).
Charybdotoxin was purchased from Alomone labs (Jerusalem, Israel),
forskolin, PMA, calphostin C, and caged IP3 were
from Calbiochem (San Diego, Calif.), 1-EBIO from Tocris (Biotrend,
Cologne, Germany); all other chemicals were purchased from Sigma
(Deisenhofen, Germany).
RNA Isolation and RNase protection assay
RNA isolation was performed following published procedures
(11)
. RNase protection assays were carried out according
to Werner et al. (12)
. Briefly, DNA probes were cloned
into the transcription vector pBluescript KSII (+) (Stratagene, San
Diego, Calif.) and linearized. An antisense transcript was synthesized
in vitro using T3 or T7 RNA polymerase and
32P-UTP (800 Ci/mmol). Samples of 20 µg RNA
were hybridized at 42°C overnight with 100,000 cpm of the labeled
antisense transcript. Hybrids were digested with RNase A and T1 for
1 h at 30°C. Protected fragments were separated on 5%
acrylamide/8M urea gels and analyzed by autoradiography. A riboprobe
complementary to nucleotides 740-1004 (AF 000 972) was used as a probe
for the detection of hSK4 mRNA. A riboprobe complementary to
nucleotides 1568–1946 (NM_002564) was used as a probe for the
detection of P2Y2 mRNA.
RT-PCR
To determine the expression of
Ca2+-activated ion channels, 5 µg total
cellular RNA from HaCaT keratinocytes or from human intestine was
reverse transcribed using 100 U Superscript reverse transcriptase
(Gibco BRL) and oligo d(T)12–17 as a primer. cDNA
fragments were amplified by PCR. We performed 30 cycles
consisting of denaturation at 95°C for 1 min, annealing at 60°C and
extension at 72°C for 1 min. The following fragments were amplified:
CLCA1, a 199 bp fragment corresponding to nt 308–506 (AF 127036,
NM_001285) using 5'-GCTGATGTTCTGGTTGCTGA-3' as a 5'-primer and
5'-CGTCAAATACTCCCCATCGT-3' as a 3'-primer; CaCC2, a 200 bp fragment
corresponding to nt 1715–1914 (AF 127035) using
5'-GGCACTTGGGCATACAATCT-3' as a 5'-primer and
5'-ACATTGGCTCCAAGAACAGG-3' as a 3'-primer; CaCC3 (CLCA2), a 202 bp
fragment corresponding to nt 2354–2555 (AF 127980, NM_006536) using
5'-GGACAGCACCTGGAGAAGAC-3' as a 5'-primer and
5'-GGCTGATGTTCAGGTCCATT-3' as a 3'-primer; hSK4, a 191 bp fragment
corresponding to nt 703–893 (AF 000972) using
5'-GGGCACCTTTCAGACACACT-3' as a 5'-primer and
5'-ACGTGCTTCTCTGCCTTGTT-3' as a 3'-primer. The amplified fragments were
separated on a 1% agarose gel and visualized by ethidium bromide
staining. As a negative control, we performed PCR with RNA that had not
been reverse transcribed.
Immunocytochemistry
Cells were washed twice with ice-cold phosphate-buffered saline
(PBS) and fixed in acetone/methanol 1:1 for 20 min at -20°C.
Endogenous peroxidases were blocked with 1%
H2O2 at room temperature.
After blocking of unspecific binding sites with 3% bovine serum
albumin (BSA)/PBS, cells were incubated overnight with a 1:500 dilution
of a K10-specific antibody (DAKO, Carpinteria, Calif.) in 3% BSA/PBS
at 4°C and rinsed three times with PBS and once with PBS/3% BSA.
After a 2 h incubation with a peroxidase-coupled anti mouse
antibody at room temperature, cells were washed three times with PBS,
once with ddH2O, and stained with the peroxidase
substrate kit using 3-amino-9-ethylcarbazole as a chromogenic substrate
(Vector Laboratories, Burlingame, Calif.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Most intriguingly, concentrations of ATP 
10 µM
were capable of producing a membrane hyperpolarization that outlasted
the drug application for at least 5 min, with some keratinocytes
remaining hyperpolarized for the rest of the observation period (up to
20 min) (Fig. 1A
). In contrast, the hyperpolarizing response
to 1 µM ATP was quickly reversible after drug washout
(n=9). If administered at 10 µM, ATP depolarized
keratinocytes by 16 ± 1 mV and then shifted the membrane voltage
by 30 ± 2 mV (relative to rest) in the hyperpolarizing direction
(n=28). A quantitatively and kinetically almost identical
response was obtained when keratinocytes were exposed to either
bradykinin (10 µM, n=3) or histamine (100 µM,
n=4), suggesting that these two substances converge onto the
same effector system that is recruited by ATP (Fig. 1B
).
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Since all three extracellular mediators are known to stimulate
phosphoinositide metabolism (13
14
15)
, we explored whether
formation of IP3 and subsequent intracellular
Ca2+ release are part of the signaling cascade.
For this purpose, whole-cell recordings were performed in which
keratinocytes were loaded with caged IP3 (10–100
µM). In all cells examined (n=7), the sudden
transformation of caged IP3 into its active form
by means of flash photolysis caused an immediate, transient
depolarization, followed by a hyperpolarizing voltage deviation (Fig. 1C
). Apart from its smaller size, the
IP3-induced biphasic voltage response closely
matched that observed after application of 1 µM ATP (Fig. 1A
). The striking similarity between the responses produced
by the three agonists and uncaged IP3 strongly
suggested that, in both cases, the biphasic change in membrane voltage
results from mobilization of Ca2+ from
IP3-sensitive stores.
Since the three extracellular mediators might also trigger signaling cascades other than the IP3 pathway, we investigated whether stimulation of adenylyl cyclase (AC) or protein kinase C (PKC) would mimic or influence the voltage response to ATP. Pretreatment of keratinocytes with the AC activator forskolin (10 µM) for 3–10 min did not affect the resting potential, nor did it alter the voltage trajectory during ATP application (n=5, data not shown). Stimulation of PKC with the phorbol ester PMA (60 ng/ml) produced a slow membrane depolarization to -36 ± 2 mV but did not modify the effect of ATP (n=4). Vice versa, inhibition of PKC with calphostin C (50 nM) caused a modest membrane hyperpolarization to -58 ± 3 mV, but again failed to influence the voltage response to ATP (n=3, data not shown).
To identify the purinoceptor subtype involved in the
electrophysiological action of ATP, we tested a series of agonists at
P1 and P2 receptors. The potency of these compounds (applied at 10
µM) to elicit a biphasic voltage response was determined as: ATP = UTP = ITP >> ADP = adenosine = 2-MeSATP >
GTP = 
,ß-meATP = Ap4A = 0 (Fig. 2A
). The lacking or negligible effect of adenosine and
,ß-meATP excluded an involvement of P1 and ionotropic P2X
receptors, respectively. The equal potency of ATP and UTP together with
the much weaker effect of 2-MeSATP and ADP indicated that the
electrophysiological action of ATP is mediated by the metabotropic P2Y2
receptor (16)
. An involvement of P2Y4 receptor, which
shares several pharmacological features with P2Y2 receptor, can be
ruled out because 1) the human P2Y4 receptor, unlike its rat
homologue, is highly selective for UTP over ATP (16)
, and
2) the P2Y4 receptor displays a very restricted distribution
in human tissues, where it is almost exclusively expressed in placenta
with low levels of expression in lung but absent in most other tissues
(16)
. Since the pharmacological profile pointed to the
P2Y2 receptor as the most likely candidate underlying the biphasic
voltage response to ATP, we used RNase protection assay to obtain
direct evidence for the expression of this purinoceptor subtype in
HaCaT keratinocytes. As shown in Fig. 2B
, HaCaT
keratinocytes indeed express P2Y2 receptor mRNA. The expression of P2Y2
mRNA was affected by the growth properties of keratinocytes, with
strong signals obtained only from proliferating keratinocytes, whereas
long-term culture of confluent keratinocytes, leading to the appearance
of the early differentiation marker K10 (cf. Fig. 5C
),
appears to coincide with the down-regulation of receptor expression.
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The strong electrical coupling of keratinocytes via gap junctions
(17)
precluded a voltage-clamp analysis of the ion
currents mediating the potential changes during ATP application. We
therefore used ion substitution and channel blockers to identify the
ion currents modulated by ATP. To test the hypothesis that activation
by ATP of Cl- channels is involved in the
transient membrane depolarization, keratinocytes were superfused with a
low Cl- solution (see Materials and Methods).
This caused an appreciable membrane hyperpolarization, possibly
reflecting the dependence of constitutively active
Cl- currents on extracellular
Cl- or the altered operation of
Cl--associated membrane transporters. If applied
to keratinocytes maintained in low Cl-, ATP (10
µM) produced a significantly stronger membrane depolarization,
(
Vm 47±8, n=7) compared to control conditions
(16±1, n=28; Fig. 3B
, P<0.001). The effects of the
Cl- channel blockers niflumic acid (100 µM,
n=8) and DIDS (50 µM, n=3) lent further support
to the notion that Cl- currents play an
essential role in the ATP-mediated membrane depolarization. Both
blockers alone exerted a hyperpolarizing action, indicating that
constitutively active Cl- conductances keep the
resting membrane potential of HaCaT keratinocytes at a depolarized
level. Despite the strongly increased driving force for depolarizing
currents in the presence of niflumic acid and DIDS, application of ATP
(10 µM) produced no or only a small depolarizing response (Fig. 3C
, 3D
). Given the importance of
Cl- channels for the depolarizing action of ATP,
we performed a set of experiments in which we used gramicidin instead
of nystatin to perform perforated patch recordings. Whereas pores
formed by nystatin display a moderate anion permeability
(PNa/PCl 10)
(18)
, gramicidin channels are completely impermeable to
Cl- and thus allowed voltage recordings without
disturbing the physiological cytosolic Cl-
concentration of our preparation (19)
. Since perforated
patch recordings with gramicidin (n=4) yielded virtually
identical results (data not shown), the depolarizing response to ATP
should represent a biological phenomenon, suggesting that keratinocytes
maintain a high intracellular Cl- concentration.
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In contrast to the effects of Cl- channel
blockers, suppression of cation currents did not inhibit the
ATP-induced depolarization to a similar extent. Replacement of external
Na+ with NMDG or the addition of
Ni2+, which blocks cation channels of
keratinocytes (20)
, to the bathing solution hyperpolarized
the membrane potential but did not abrogate the depolarizing response
(Vm) to ATP, which was 11 ± 2 mV (n=4) and
13 ± 3 mV (n=4) in NMDG- and
Ni2+-containing bath solution, respectively (Fig. 3E
, 3F
). Although these responses were not
significantly different from control in terms of the Vm
values, one should bear in mind that they were evoked from a
substantially more negative membrane potential compared to control
conditions. The membrane hyperpolarization enhances the driving force
for inward current flow through cation channels and reduces outward
current flow through hyperpolarizing K+ channels.
It follows that if the Cl- conductance was the
only source of the depolarizing response to ATP, Vm should
have been substantially larger in the presence of the hyperpolarizing
agents NMDG or Ni2+. Since Vm remained below
this expected value, we conclude that activation of cation currents
contributes to the depolarizing effect of ATP.
We next identified the ion conductance mediating the prominent and
long-lasting hyperpolarization. Assuming that K+
currents were the most likely candidates, we measured the effect of ATP
in the presence of several K+ channel blockers.
Charybdotoxin (ChTx) produced a dose-dependent inhibition of the
hyperpolarizing response to ATP. As shown in Fig. 4A
, ChTx (10–100 nM, n=15) drastically slowed
repolarization of the initial depolarization and prevented the strong
hyperpolarization observed under control conditions. If applied at 100
nM (n=4), ChTx completely abolished the repolarizing
component of the ATP response, so that the membrane potential stayed on
a depolarized plateau for the entire period of ATP application (Fig. 4A
). The same effect was obtained with 1 µM clotrimazole
(n=3, Fig. 4B
). If keratinocytes were exposed to
ChTx (100 nM) after the hyperpolarizing effect of ATP had fully
developed, a complete reversion of the hyperpolarization was obtained,
despite the continued presence of ATP (n=2; Fig. 4C
). None of the other K+ channel
blockers tested (TEA, Ba2+, and verapamil) gave
rise to a similar plateau depolarization during superfusion with ATP.
Nevertheless, both TEA (20 mM) and Ba2+ (2 mM)
reduced the extent of the ATP-evoked hyperpolarization without
appreciably slowing its kinetics. The most negative membrane potentials
attained under these conditions were -68 ± 2 mV (n=4)
and -57 ± 3 mV, (n=4), respectively, but only the
hyperpolarization in the presence of BaCl2 was
significantly smaller compared to control (P<0.001).
Verapamil (10–100 µM), which had been previously shown to block some
K+ currents of keratinocytes (6)
,
did not affect the hyperpolarizing action of ATP (hyperpolarization to
-73±1 mV, n=3). The high sensitivity of the ATP-induced
hyperpolarization to ChTx and clotrimazole was consistent with the
activation of a voltage-independent,
Ca2+-activated K+ current.
Since the ion channels conducting this current appear to be mainly
closed under control conditions (i.e., in the absence of extracellular
signaling molecules), we investigated whether 1-EBIO, an activator of
epithelial, Ca2+-activated
K+ channels (21)
, was capable of
mimicking the hyperpolarizing action of ATP. As shown in Fig. 4D
, 1-EBIO (1 mM) indeed produced a rapid and prominent
hyperpolarization to -83 ± 3 mV (n=5). If applied in
the presence of 1-EBIO, ATP (10 µM) gave rise to the typical
transient depolarization, which was, however, much larger than under
control conditions (cf. Fig. 1A
), reflecting the enhanced
driving force for Cl- and cation currents (Fig. 4D
).
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Since both the flash photolysis of caged IP3 and
the pharmacological experiments strongly favored
Ca2+-activated ion currents as effectors of the
ATP-triggered signaling cascade, we performed RT-PCR with primers
specific for sequences of three Cl- channels,
CaCC2, CaCC3 (CLCA2), and CLCA1, and one K+
channel, hSK4. We obtained positive signals for two
Cl- channels, CaCC2 and CaCC3(CLCA2), whereas
the third Cl- channel examined (CLCA1) was not
expressed in HaCaT keratinocytes. Furthermore, our cells were found to
express the Ca2+-activated
K+ channel hSK4 (Fig. 5A
).
Does the powerful hyperpolarizing effect of hSK4 activation bear
significance on proliferation and differentiation of keratinocytes? To
address this question, RNase protection assays were performed using RNA
from proliferating, subconfluent HaCaT keratinocytes from cells that
had just reached confluence and from quiescent, postconfluent cells (4
days after they had reached confluence). As illustrated in Fig. 5B
, high levels of hSK4 mRNA were detected in exponentially
growing cultures and immediately after the keratinocytes had reached
confluence. By contrast, expression of hSK4 mRNA was markedly reduced
in postconfluent cells that had undergone partial differentiation, as
reflected by the presence of a large number of cells expressing the
differentiation-specific keratin 10 (K10, Fig. 5C
)
(22
, 23)
. These results demonstrate that the onset of
HaCaT cell differentiation is associated with a striking
down-regulation of hSK4 mRNA expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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14
15
, 24
25
26)
. Based on its pharmacology and expression
in keratinocytes, P2Y2 was identified as the purinoceptor subtype
mediating the electrophysiological response to ATP in these cells. The
receptor subtypes interacting with bradykinin and histamine were not
investigated here.
Electrophysiological studies reported that keratinocytes possess both
Ca2+-activated Cl- and
Ca2+-activated cation channels (1
, 2
, 5)
. Since suppression of Cl- channels
inhibited the depolarizing response much stronger than suppression of
cation channels, activation of a Ca2+-dependent
Cl- conductance emerges as the predominant
mechanism of the mediator-induced depolarization, whereas the
contribution of Ca2+-activated cation currents
seems smaller. The involvement of a
Ca2+-activated Cl- channel
in the membrane depolarization is further corroborated by the blocking
action of niflumic acid, which is known to suppress
Ca2+-activated Cl-
channels (27)
. Consistent with our data, bradykinin was
reported to use the same transduction pathway, i.e.,
Ca2+i mobilization and
subsequent activation of a Ca2+-dependent
Cl- conductance, to depolarize the membrane
potential of NRK fibroblasts (28)
. Since ATP gives rise to
a plateau depolarization when K+ channels are
blocked, deactivation of the Cl- conductance is
unlikely to account for the repolarizing phase of the depolarization.
Rather, repolarization as well as subsequent hyperpolarization must
result from activation of a K+ conductance that
overcomes the depolarizing action of the Cl-
conductance.
Because manipulations of cAMP- or PKC-dependent signal transduction
processes failed to influence the electrophysiological effects of the
mediators, we propose that the IP3-mediated
release of Ca2+i is the central
signaling pathway leading to the activation of
Cl-, K+, and, to a lesser
extent, cation channels. In agreement with our findings, activation by
extracellular ATP of Ca2+-sensitive
K+ and Cl- channels via
mobilization of Ca2+i was
reported from several preparations, including distal nephron epithelial
cells, coronary artery smooth muscle, endometrial epithelium, pulmonary
artery endothelial cells, and bronchial epithelial cells
(29
30
31
32
33)
. A salient feature of the electrophysiological
action of ATP, which to our knowledge has not been reported before, is
its ability to induce a prolonged, self-sustained hyperpolarization
after drug washout. Since this voltage behavior was only observed at
ATP concentrations 10 µM, whereas 1 µM ATP produced a
rapidly reversible hyperpolarization, it appears likely that the
ATP-induced rise in Ca2+i has to
exceed a threshold concentration to exert a long-term effect on the
electrophysiological properties of keratinocytes. The dose-response
curve for ATP to changes in
Ca2+i of keratinocytes lends
support to this hypothesis: Whereas 1 µM ATP enhances
Ca2+i by 40%, 10 µM and 100
µM ATP increase Ca2+i by 280%
and 350%, respectively (14)
. It is tempting to speculate
that at low ATP concentration, the hyperpolarizing voltage trajectory
follows the time course of cytosolic Ca2+ release
(14
, 25)
, whereas at higher ATP concentrations, an
additional pathway is recruited that promotes opening of
K+ channels.
Recently, the first members of the Ca2+-dependent
Cl- channel family in humans were cloned. This
family is termed hCaCC or hCLCA, with hCaCC1 and hCaCC3 being identical
to hCLCA1 and hCLCA2, respectively (34
35
36)
. In
situ hybridization studies of mouse skin revealed a murine
Ca2+-activated Cl- channel
homologue (mCaCC) in epidermal keratinocytes (37)
, but the
presence of hCaCCs in human skin had not been determined yet. We report
here that HaCaT keratinocytes express both hCaCC2 and hCaCC3, but not
hCaCC1. The absence of the latter agrees well with a previous study
demonstrating selective expression of hCaCC1 in a subset of human
intestinal epithelial cells (34)
. In contrast, the
distribution pattern of hCaCC2 and hCaCC3, although also highly
restricted, comprises several types of tissue (36)
.
Expression of hCLA2/hCaCC3 in HEK-293 cells gave rise to a
Ca2+-dependent Cl- current
that was sensitive to both niflumic acid and DIDS (35)
. It
thus appears safe to conclude that hCaCC2 and hCaCC3 are the most
likely candidates to mediate the ATP-induced,
Ca2+-activated Cl-
conductance observed in our preparation.
Our study is the first to demonstrate expression of hSK4 (also termed
hIK or hIK1) in human keratinocytes. It has been disputed whether this
channel belongs to small conductance,
Ca2+-activated K+ channels
(SK) or whether it is a representative of intermediate conductance,
Ca2+-activated K+ channels
(IK) (9
, 38
39
40)
. Its exclusive expression in nonexcitable
tissue would argue in favor of hSK4 belonging to IK, which, in contrast
to SK, apparently is absent in excitable cells but present in various
blood cells and endothelial as well as epithelial cells of several
organ systems (38)
. A functional hallmark of IK is its
strong activation by intracellularly released
Ca2+ evoked by extracellular mediators such as
ATP, bradykinin, and histamine (9
, 38
39
40)
. Expression of
hSK4/hIK in CHO cells or HEK-293 cells yielded a
K+ current with the same pharmacological features
as the ATP-induced membrane hyperpolarization reported here from
keratinocytes: functionally expressed hSK4 channels were highly
sensitive to ChTx and clotrimazole, but weakly sensitive to TEA
(38
, 39)
. In addition, they were activated by 1-EBIO
(39)
. Although we cannot exclude the contribution of
other, hitherto undetected Ca2+-activated
K+ channels, it is very likely that, at minimum,
a large portion of the ATP-induced membrane hyperpolarization in our
preparation is conducted by hSK4.
That hSK4 plays an important role for cellular events associated with
proliferation and/or differentiation of keratinocytes is suggested by
the observation that its expression is tightly regulated by the culture
conditions. Whereas strong hSK4 signals were obtained in proliferating
keratinocytes, only a weak signal was found in differentiating
keratinocytes. An inverse expression pattern was reported by Mauro et
al. (6)
for a K+ channel with a much
higher unitary conductance (70 pS) than hSK4 channels (typically 12–30
pS) and a different pharmacological profile. In contrast to hSK4, this
type of K+ channel was only detected in
differentiated keratinocytes, where it is thought to be involved in
Ca2+-induced differentiation (6)
. We
thus propose that hSK4 serves primarily to mediate agonist-induced
membrane hyperpolarization during cell proliferation when resting
membrane potential is relatively depolarized, whereas the 70 pS
K+ channel seems to be important for
differentiated cells to maintain their resting membrane potential at
more negative values.
Given that hSK4 appears to be an important target of P2Y2 receptor
activation, it is noteworthy that the expression of P2Y2 transcripts
declined in a fashion similar to that of hSK4 transcripts, suggesting
that the expression of receptor and effector are closely coupled in
keratinocytes. Underscoring the importance of P2Y2 receptors in
keratinocyte proliferation, in situ hybridization studies of
human skin sections showed a striking epidermal gradient of the
localization of P2Y2 transcripts, with strong signals being confined to
actively proliferating basal cells, whereas only weak signals were
detected in outer, more differentiated cell layers (26)
.
This agrees well with experiments showing that ATP promotes
proliferation and inhibits differentiation of keratinocytes (14
, 26
; but see ref 41
). Since in our hands ATP is
effective at concentrations as low as 1 µM, it is attractive to
postulate that the electrophysiological actions reported here bear
significance on the behavior of keratinocytes in intact or damaged
epidermis. For example, in wounded skin, keratinocytes are exposed to
10–30 µM ATP released from activated platelets (42)
,
which should be an important signal for their proliferation.
An additional line of evidence assigning biological significance to the hSK4-mediated hyperpolarization comes from our preliminary observation that the strong activation by 1-EBIO of hSK4 kept HaCaT keratinocytes in an undifferentiated state, as indicated by the absence of K10 immunostaining typically present in control cells maintained under the same conditions. These data lend further support to the notion that the expression of hSK4 is intimately associated with the proliferation of keratinocytes and that the disappearance of hSK4 coincides with the inhibition of proliferation and subsequent differentiation. We thus propose that the expression and regulation of hSK4 serves as a central interface linking the electrophysiological properties of keratinocytes to their growth and differentiation pattern. Experiments using a conditional down-regulation of hSK4 by antisense approach or data from tissue-specific knockout experiments should help to further elucidate the role of this ion channel in the proliferation and differentiation of keratinocytes.
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ACKNOWLEDGMENTS |
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Received for publication April 5, 2000.
Revision received June 8, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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