KCNA10: a novel ion channel functionally related to both
voltage-gated potassium and CNG cation channels
Rainer
Lang1,*,
George
Lee2,*,
Weimin
Liu2,
Shulan
Tian2,
Hamid
Rafi2,
Marcelo
Orias2,
Alan S.
Segal1, and
Gary V.
Desir2
2 Yale University School of Medicine and West
Haven Veterans Affairs Medical Center, New Haven, Connecticut
06510; and 1 University of Vermont, Burlington,
Vermont 05446
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ABSTRACT |
Our laboratory previously cloned a novel rabbit gene
(Kcn1), expressed in kidney, heart, and aorta, and predicted to
encode a protein with 58% amino acid identity with the K channel
Shaker Kv1.3 (Yao X et al. Proc Natl Acad Sci USA 92:
11711-11715, 1995). Because Kcn1 did not express well
(peak current in Xenopus laevis oocytes of 0.3 µA at +60 mV),
the human homolog (KCNA10) was isolated, and its expression was
optimized in oocytes. KCNA10 mediates voltage-gated K+
currents that exhibit minimal steady-state inactivation. Ensemble currents of 5-10 µA at +40 mV were consistently recorded from injected oocytes. Channels are closed at the holding potential of
80 mV but are progressively activated by depolarizations more positive than
30 mV, with half-activation at +3.5 ± 2.5 mV.
The channel displays an unusual inhibitor profile because, in addition to being blocked by classical K channel blockers (barium
tetraethylammonium and 4-aminopyridine), it is also sensitive to
inhibitors of cyclic nucleotide-gated (CNG) cation channels (verapamil
and pimozide). Tail-current analysis shows a reversal potential shift
of 47 mV/decade change in K concentration, indicating a K-to-Na
selectivity ratio of at least 15:1. The phorbol ester phorbol
12-myristate 13-acetate, an activator of protein kinase C, inhibited
whole cell current by 42%. Analysis of single-channel
currents reveals a conductance of ~11 pS. We conclude KCNA10 is a
novel human voltage-gated K channel with features common to both
K-selective and CNG cation channels. Given its distribution in renal
blood vessels and heart, we speculate that KCNA10 may be involved in
regulating the tone of renal vascular smooth muscle and may also
participate in the cardiac action potential.
potassium channel; patch clamp; cyclic nucleotide voltage-gated; vasoregulation; Xenopus laevis oocyte; human
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INTRODUCTION |
POTASSIUM CHANNELS, a diverse family of
membrane proteins, play a pivotal role in regulating membrane potential
and contribute to many vital cellular processes. Previously, our
laboratory cloned a novel rabbit K channel gene
(Kcn1) that is related to the Shaker superfamily. The protein encoded by the longest open reading frame has
58% amino acid identity with Kv1.3 (20), and its predicted secondary
structure is identical to that of Shaker-related K channels, including intracellular NH2 and COOH termini, six
transmembrane segments, a voltage sensor (S4), and a pore (P)
region. Unlike Shaker proteins, however, Kcn1
contains a putative cyclic nucleotide (CN)-binding domain at the COOH
terminus, suggesting that protein function could potentially be
regulated by cyclic nucleotides. Kcn1, therefore, may be a
member of a new class of ion channels, which includes HERG (16), Eag
(3, 7), and ATK1 (15), and BCNG-1 (14) with structural features common
to both voltage-gated Shaker-like K channels and cyclic
nucleotide-gated (CNG) nonselective cation channels.
These proteins may play important roles in mediating the effects of
substances, such as nitric oxide (NO), that increase intracellular cyclic nucleotides. For example, in rat pulmonary artery rings, cGMP-dependent but not -independent relaxation was inhibited by the
classic K channel blockers tetraethylammonium (TEA) and charybdotoxin (CTX) (2). Furthermore, in whole cell patch-clamp studies, NO and cGMP
increased whole cell K current, suggesting that the final common
pathway shared by NO and the nitrovasodilators is cGMP-dependent K
channel activation (2). This new class of channels may
also participate in hormone secretion. It was recently demonstrated
that adrenocorticotopic hormone (ACTH) regulates cortisol
synthesis/secretion by adrenal zona fasciculata cells by inhibiting a K
current (Iac) that combines features of
voltage-gated K channels with those of CNG nonselective cation channels
(6, 18). Finally, it has been suggested that some members of this class
of ion channels modulate membrane excitability in the brain. Indeed,
BCNG-1, a novel, brain-specific ion channel protein isolated by
interactive cloning with the SH3 domain of N-src, contains a
well-conserved CNG-binding domain (14).
Kcn1 gene expression, assessed by RNAse protection assay and
PCR of dissected nephron segments, was strongest in heart, aorta, and
renal blood vessels (19, 20). When expressed in Xenopus laevis
oocytes, Kcn1 yielded small currents (0.2-0.4 µA); thus we were unable to accurately assess its kinetic and pharmacological properties. The clone expressed poorly either because its message was
unstable and/or inefficiently translated in oocytes, or that an
additional subunit was required. The human homolog of Kcn1 (KCNA10) was cloned by library screening (12). KCNA10 is 90% identical
to Kcn1 at the amino acid level, and its secondary structure is
the same, including a putative cGMP-binding domain at the COOH terminus. Expression of KCNA10 was optimized in oocytes such that it is
now possible to consistently record whole cell currents of 5-10
µA at +40 mV. This paper describes the basic kinetic and pharmacological properties of the channel.
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MATERIALS AND METHODS |
Cloning of KCNA10 cDNA in High-Expression Vector
The coding region of KCNA10 (accession no. U96110) was amplified by PCR
with a proofreading polymerase and a sense primer containing a
BamH 1 site and an antisense primer with a Xba
1 site: sense, 5'CGGGGATCCCTCCCCTAGAATGGATGTG3' ;
antisense, 5' TCTAGAAAGAGACAGGATGGACCCAAGAAGCC3'. The
amplified product was cut with BamH 1 and Xba 1 and
ligated into PGH19, a high-expression vector, on the basis of pGEM
(Promega, Madison, WI), containing 60 bp of the 5'UTR of X. laevis
-globin and 300 bp of the 3'UTR. The construct was
sequenced by the dideoxynucleotide chain termination method of Sanger
et al. (13) to ensure that mutations were not introduced during
amplification. Nucleotide and protein sequence analyses were carried
out by using MacVector (Oxford Molecular Group PLC) and Lasergene
(DNASTAR, Madison, WI). cRNA was synthesized with T7 after
linearization with Not1.
Expression of KCNA10 in X. Laevis Oocytes
Oocyte preparation.
Stage V-VI oocytes from X. laevis were harvested from
ovarian lobes and repeatedly washed in Ca2+-free OR2 (in
mM: 82.5 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES, pH 7.5). Oocytes were defolliculated by incubation with type IA collagenase (2 mg/ml) in Ca2+-free OR2 for 90-120 min at room
temperature. After a second washing with Ca2+ -free OR2,
the oocytes were stored in ND96 (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, and 5 HEPES, pH 7.5) with
penicillin (100 U/ml)/streptomycin (100 µg/ml), and sodium pyruvate
(550 mg/l) at 19°C. The vegetal poles of selected oocytes were
injected with either 50 ng of 5' capped KCNA10 cRNA or RNAse-free
water as control.
Two-microelectrode voltage clamp.
Whole cell currents were recorded by using standard two-microelectrode
voltage clamp. Current measurements were made after a 3- to 14-day
incubation period, and expressed potassium currents were compared with
those from water-injected control oocytes. For voltage clamping,
oocytes were impaled with two microelectrodes filled with 1 M KCl
(resistance 0.5-2 M
). Oocyte current-voltage (I-V) relationships were obtained by applying command
voltage-ramp or step protocols and measuring the resulting membrane
current with a current-to-voltage amplifier (OC-725, Warner
Instruments, Hamden, CT). Data acquisition was controlled
by either PULSE 8.1 (Heka Elektronik) or pClamp 5.5 (Axon Instruments,
Foster City, CA). Currents were filtered at 1-2 kHz and digitized
to hard disk at 2.5-5 ksamples/s. Data were analyzed by using
PULSE-Fit (Heka Elektronik), Origin 5.0 (Microcal Software,
Northampton, MA), and Igor-Pro (WaveMetrics, Lake Oswego, OR). The
extracellular recording solution was ND96 unless otherwise noted. For
ion-substitution experiments, the chloride-salt of the test cation was
substituted for NaCl, and ion selectivity was measured by using
tail-current analysis.
Patch clamp.
To prepare cells for patch clamping, oocytes were placed in a
hypertonic K-aspartate solution so that the vitelline membrane could be
removed manually by using fine forceps. Devitellinized oocytes were
placed in a recording chamber containing ND96 (Na-ND96 or K-ND96) bath.
Patch pipettes were filled with Na-MES (2 mM K+) and had
resistances of 4-8 M
. Single KCNA10 channel currents in the
cell-attached and inside-out configuration were amplified with an EPC-9
computer-controlled patch-clamp amplifier (Heka Elektronik). For
display, currents were filtered at 400 Hz and records were analyzed by
using the programs mentioned above and software written in our
laboratory (1). All chemicals were purchased from Sigma Chemical (St.
Louis, MO). ShK-Dap22 was a generous gift from Dr. Mike
Pennington (Bachem).
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RESULTS AND DISCUSSION |
KCNA10 is a Voltage-Gated K Channel
As noted, KCNA10 contains regions homologous to the voltage sensor of
Shaker-like K channels; so we first tested the hypothesis that
KCNA10 is a voltage-gated channel. Figure
1A shows representative ensemble
currents indicating that KCNA10 behaves as a voltage-gated channel when
expressed in an oocyte bathed in Na-ND96. From a holding potential of
80 mV, successive depolarizing steps (from
60 to +40 mV,
see Fig. 1, inset) reveal voltage-gated outward currents
elicited beyond a threshold voltage of about
30 mV. Replacing
bath Cl
with an impermeant anion such as gluconate
does not affect recorded currents (data not shown), indicating that the
outward current is carried by K+. KCNA10 current exhibits
minimal steady-state inactivation. Figure 1B shows the
I-V curve resulting from this voltage protocol. The voltage for
half-maximal activation (V1/2) was obtained by
fitting the resultant conductances to the Boltzmann equation. A
representative conductance-voltage (G-V) plot (Fig.
1C, n = 6 cells) shows that the
V1/2 is about +3.6 mV. The maximal conductance
occurs around +40 mV. To estimate the cation selectivity of KCNA10,
tail currents from +30 mV were examined at voltages from
100 to
+20 mV. A plot of reversal potential vs. bath K concentration
([K]) is shown in Fig. 2. The
zero-current K-to-Na permeability ratio, based on a linear fit of the
observed change in reversal potential per decade change in external
[K], is PK/PNa = 15. Substitution
of gluconate for chloride did not change the magnitude of the outward current. These results indicate that KCNA10 expresses a highly selective voltage-gated K channel. They are consistent with the fact
that KCNA10 has a voltage gate (S4) and a pore region that are nearly
identical to those of Shaker voltage-gated K channels.


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Fig. 1.
KCNA10 is a voltage-gated K channel. A: whole cell KCNA10
currents recorded from a Xenopus laevis oocyte under
2-electrode voltage clamp, showing that KCNA10 behaves as a
voltage-gated channel with a threshold voltage around 10 mV.
Note that ensemble KCNA10 currents exhibit minimal steady-state
inactivation. Inset: voltage protocol with holding potential of
80 mV and test pulses from 60 to +50 mV in 10-mV steps.
B: current-voltage (I-V) relationship for
ensemble KCNA10 currents subjected to voltage protocol shown in
A. C: whole cell normalized conductance-voltage
(G-V) curve for hKCNA10. The G-V plot
from 6 cells and their mean (fit by a Boltzmann function) are shown.
The half-maximal conductance occurs at half-maximal activation
(V1/2) = +3.64 mV. Gmax,
maximal G; Vcommand, command voltage.
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Fig. 2.
KCNA10 is K selective. Selectivity for K+ compared with
Na+ was assessed by applying the tail-analysis voltage
protocol (inset) to expressing oocytes in varying bath K
concentrations ([K]). Slope of linear fit of data is 47 mV/decade (a perfect K electrode would have a slope of 58 mV/decade),
indicating that KCNA10 prefers K+ to Na+ by at
least 15:1.
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Activation Parameters and Ionic Selectivity
Peak current was 5.5 ± 1.5 µA (n = 30) at a membrane
potential of +40 mV, and time-dependent inactivation was not observed. Activation parameters were determined by using the Pulse-Fit program (Heka), and the observed currents were fitted to a variant of the
Hodgkin-Huxley equation. The time constant of activation
(
act) was determined by using voltage steps from
60 to +40 mV from a holding potential of
80 mV. An
example of an activation curve (from
80 to +40 mV) fit by an
exponential is shown in Fig. 3A, whereas Fig. 3B shows
act as a function of
voltage. In Fig. 3B (left), we now show the
act vs. voltage from the raw data fit with single
exponentials. From Fig. 3 it may be seen that
act approaches 20 ms at the most depolarized voltages. In Fig. 3B (right), these data are normalized and fit with the mean single exponential, which shows that the average "voltage constant" for the pooled data is 16.0 ± 0.5 mV. This voltage constant indicates that
act decreases e-fold for each 16 mV of
depolarization. Compared with other Kv channels, KCNA10 activates
relatively slowly (18 ms for KCNA10 vs. 2 ms for Kv1.5, Table
1). Although outward current was voltage
gated, compared with most Kv channels the threshold voltage for
activation of KCNA10 is shifted to the right (Table 1). Indeed,
currents were not observed at voltages more hyperpolarized than
20 mV. It should be noted that these results reflect the kinetic
behavior of the
-subunit alone. Presently, there are no data
supporting the existence of additional subunits. However, because many
Kv channels have been shown to be heteromultimeric in vivo, it is
likely that KCNA10 also interacts with an
-subunit, which might
modulate its kinetic behavior.


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Fig. 3.
Activation of KCNA10 is voltage dependent. A: whole cell
activation curve for hKCNA10 fit by a single exponential. In this
example, voltage is stepped from 80 to +40 mV, resulting in a
nearly exponential rise [time constant of activation
( act) = 13 ms] of voltage-gated current toward a
steady-state current of 9 µA. B: act vs.
voltage for hKCNA10 measured in 6 oocytes (left). Points for
each cell are fit with a single exponential. Right: raw data
have been normalized, and mean single exponential fit (dashed curve)
shows a mean "voltage constant" of 16.0 ± 0.5 mV (n = 6), indicating that act decreases e-fold with
every 16 mV of depolarization. At strong depolarizations,
act approaches 20 ms.
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Inhibitor Profile
K channel blockers with broad selectivity [barium,
4-aminopyridine (4-AP) and TEA] when added to the bath solution
all resulted in reversible inhibition of KCNA10 currents (Table
2). The inhibitory effects of barium and
4-AP occurred within 30 s, whereas maximal inhibition by TEA took up to
15 min. At a voltage of +40 mV, 2 mM 4-AP decreased current by 64.6 ± 5.8% (n = 7), whereas 5 mM barium inhibited it by 47.7 ± 10.9% (n = 4). The dose-response curve for TEA was determined
as shown in Fig. 4. The inhibition constant
Ki for TEA was 53 mM. More selective blockers of K
channels were also tested. Application of 30 nM apamin did not affect
KCNA10 currents (data not shown). However, 200 nM of CTX (n = 5) caused an immediate and reversible blockade of outward current
(Table 2) without any change in activation kinetics.
ShK-Dap22 (mutant of ShK, a polypeptide isolated from the
sea anemone Stichodactyla helianthus) is a very selective and
potent (Ki = 50 pM) inhibitor of Kv1.3 (8).
Fifty-picomolar ShK-Dap22 had no effect on KCNA10 current.

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Fig. 4.
Dose-response curves for KCNA10. Dose-response curves for inhibitors of
K channels [tetraethylammonium (TEA), ketoconazole] and CNG
cation channels (verapamil, pimozide) were determined. Data were
obtained at +40 mV. IgorPro was used to determine best fit for data.
Single exponential fits are shown for verapamil, TEA, and ketoconazole.
A double exponential fit is shown for pimozide. Pimozide was the most
potent inhibitor of KCAN10 current.
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Although the precise physiological role of KCNA10 in the heart is not
yet known, it is expressed there and its kinetic properties would
permit it to participate in cardiac repolarization. Therefore, known
inhibitors of cardiac ion channels, such as ketoconazole, lanthanum,
and cobalt, were tested for their ability to block KCNA10 current.
Ketoconazole, a drug used to treat fungal infections, has been
associated with Q-T prolongation and torsade de pointes when given in
combination with the antihistamine terfenadine (Seldane). This is
partly due to the fact ketoconazole also uses the cytochrome P-450 metabolic pathway and causes the plasma concentration of terfenadine to increase. In addition, it was recently shown that at
concentrations within the therapeutic range, ketoconazole also blocks
two K channels, HERG (IC50 = 49 µM) and Kv1.5
(IC50 = 107 µM), known to participate in cardiac
repolarization (14). In X. laevis oocytes, 0.3 µM
ketoconazole inhibited KCNA10 current by 34 ± 2.3% (n = 11, Fig. 4), suggesting that KCNA10 is significantly more sensitive to the
action of ketoconazole than either HERG or Kv1.5. The addition of 2 mM
of cobalt blocked KCNA10 current (Fig. 5)
and affected the activation kinetics by increasing
act (Table 2). Lanthanum at a concentration of 100 µM had no effect on
KCNA10 current.

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Fig. 5.
Cobalt affects kinetics of activation. Addition of 2 mM of
cobalt to extracellular solution (n = 3) increases
act and voltage dependence of activation.
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KCNA10 has structural features common to both voltage-gated K channels
and CNG channels. Therefore, antagonists of CNG cation channels
(pimozide and verapamil) were also tested for their ability to block
KCNA10 currents. The most potent and specific blockers of CNG channels
are the diphenylbutylpiperidines including penfluridol, fluspirelene,
and pimozide. Pimozide inhibited KCNA10 current, with a
Ki of 300 nM (Table 2). Although less potent,
verapamil also inhibited KCNA10 current, with a Ki
of 53 µM (n = 5, Fig. 4). Inhibition by verapamil was
immediate. In the presence of verapamil, KCNA10 exhibited C-type
inactivation (current at 0.8 s less than peak, Fig.
6, A and B), suggesting
that the block occurs in the open configuration. Furthermore, although
peak current returned to control levels after washout, current at 0.8 s
was still less than peak (Fig. 6C), indicating that verapamil
can block KCNA10 from the cytoplasmic side. Verapamil has been reported to affect Kv1.3 similarly (10). Interestingly, Kv1.3 and KCNA10 are
located on chromosome 1 at p13.1 and are probably the result of gene
duplication (12). Taken together, these data strongly suggest that
KCNA10 has pharmacological properties common to both voltage-gated K
channels and CNG cation channels. They confirm the notion, initially
suggested by the presence of a putative cyclic nucleotide-binding
domain at the COOH terminus, that KCNA10 belongs to a novel class of K
channels that displays some of the properties classically associated
with CNG channels.

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Fig. 6.
Verapamil inhibits KCNA10 and alters its kinetics. Effect of 100 µM
verapamil on KCNA10 ensemble currents is shown in a representative
experiment. A and B: note that in addition to blocking
~50% of current amplitude, kinetics of inactivation are altered.
C: persistence of this kinetic effect after washout of drug
suggests that verapamil blocks channel from the cytoplasmic side.
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Regulation by Second Messengers
Either cAMP and/or cGMP can directly activate CNG nonselective cation
channels. The cyclic nucleotides bind to a domain located at the COOH
terminus (5) of these channels. Unlike CNG currents, the
Iac current (K-selective channel in AZF cells) is
inhibited by cAMP through a mechanism that depends on ATP hydrolysis
(8). Although the molecular identity of the Iac
channel is not yet known, it is postulated to be a heteromultimer.
Heterologous expression (X. laevis oocytes, mammalian cells) of
cloned pore-forming subunits (
) of CNG channels mediates
nonselective cation currents that are gated directly by cyclic
nucleotides (5). In contrast,
-subunits of K channels known to
contain putative cyclic nucleotide-binding domains (EAG, HERG, BCNG-1,
ATK1) do not appear to be directly regulated by cyclic nucleotides when
expressed in heterologous systems. This raises the possibility that
auxiliary (
) subunits are needed for the action of cyclic
nucleotides on the
-subunits of these channels.
To test whether cyclic nucleotides modulated KCNA10 current,
membrane-permeant forms of cGMP and cAMP [8-bromoguanosine
3',5'-cyclic monophosphate (8-BrcGMP) and 8-bromoadenosine
3',5'-cyclic monophosphate (8-BrcAMP), respectively]
were added to the oocyte bath. Baseline peak current was decreased
slightly (14.93 ± 4.33%, n = 11) by 2 mM 8-BrcGMP (Fig.
7A) and was unaffected by 2 mM
8-BrcAMP (n = 8) (Fig. 7B). Additional experiments were
also performed to examine possible effects of cyclic nucleotides. With
regard to cAMP, 1) oocytes were treated with a cocktail of
forskolin (10 mM), dibutyryl-cAMP or 8-BrcAMP (2 mM), and IBMX (100 mM); and 2) cAMP was directly applied to excised membrane
patches. Neither had any significant effect. With regard to cGMP,
1) oocytes were treated with membrane-permeant 8-BrcGMP (2 mM);
2) oocytes were treated with a cocktail of
S-nitroso-N-penicillamine (10 mM; a NO
donor) and zaprinast (100 mM; a cGMP phosphodiesterase inhibitor); 3) oocytes were directly injected with cGMP such that the
intracellular concentration was ~1 mM; and 4) cGMP was
applied directly to excised membrane patches. Again, none of these
maneuvers significantly altered the currents recorded. These data
suggest that the activity of the
-subunit of KCNA10 is not regulated
by cyclic nucleotides. These results are similar to those observed for
other K channels that contain cyclic nucleotide-binding domains,
indicating, as previously mentioned, that accessory subunits may be
required.


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Fig. 7.
Effect of cyclic nucleotides on KCNA10 current. Currents are measured
before and 20 min after membrane-permeant forms of either cGMP or cAMP
were added to bath. A: 2 mM of 8-bromoguanosine
3',5'-cyclic monophosphate (8-BrcGMP) decreased current by
14.93 ± 4.3% (n = 11) from control levels. B:
addition of 2 mM 8-bromoadenosin-3',5'-cyclic monophosphate
(8-BrcAMP) did not affect expressed current (n = 8).
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The effect of internal calcium on channel activity was also tested.
Intracellular Ca2+ concentration
([Ca2+]) was controlled by varying
extracellular [Ca2+] in presence the calcium
ionophore ionomycin. Up to 200 µM external Ca2+ did not
affect channel activity (data not shown), thereby suggesting that
KCNA10 activity is not regulated by intracellular calcium. These
results are not surprising because the predicted amino acid sequence
lacks a calcium-binding domain. However, they do not rule out the
possibility that KCNA10 activity could be regulated by calcium in vivo,
particularly if KCNA10 is a heteromultimeric complex.
Computer analysis of the amino acid sequence of KCNA10 indicates the
presence of five putative protein kinase C (PKC) phosphorylation sites.
Therefore, the effect of PKC on KCNA10 current was examined by the
addition of 10 nM phorbol ester phorbol 12-myristate 13-acetate (PMA),
an activator of PKC. Peak K current began to decline irreversibly within 5 min, and by 20 min the mean decline was 42 ± 2.8%,
(n = 5). Figure 8,
A and B, depicts a typical experiment, showing that PMA
inhibits KCNA10 current without affecting its kinetics parameters. The
effect of PMA was specific because application of
12-(1-pyrene)dodecanoic acid and 4-PMA, phorbol esters that do not
activate PKC, did not affect KCNA10 currents (Fig. 8, A and
C). These results, which were also confirmed in cell-attached macropatches, suggest that phosphorylation by PKC modulates channel activity. However, we cannot exclude the possibility that part of the
decrease in current by PMA is due to internalization of some of the
oocyte's plasma membrane (17). Inhibition of K channel activity by PKC
has been reported for other Kv channels (4, 9) and also for maxi-K
channels (11). Mutational analysis of the putative PKC sites is
presently underway to determine whether any of them mediate the
inhibitory effect observed with PMA.

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Fig. 8.
Phosphorylation by protein kinase C (PKC) reduces KCNA10 current.
A and B: addition of 10 nM phorbol 12-myristate
13-acetate (PMA), an activator of PKC, causes KCNA10 current to
decrease by an average of 42% without affecting kinetic properties of
current. Inactive analogs of PMA [e.g., 4 -PMA and
12-(1-pyrene)dodecanoic acid (PDA)] did not affect KCNA10
currents. C: I-V curves for currents shown in
A and B.
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Single KCNA10 Channels
Oocytes expressing >2 µA of voltage-gated K current at +40 mV under
two-electrode voltage clamp were subsequently devitellinized for patch
clamp experiments. Figure 9A shows
typical KCNA10 single-channel currents recorded from an inside-out
membrane patch excised into K-ND96 (96 mM K+). The patch
pipette was filled with Na-MES with 2 mM K+. From a holding
potential of
80 mV, the command voltage was stepped to the
potentials indicated to elicit voltage-gated KCNA10 currents. Under
these conditions, KCNA10 channels were apparent at
40 mV.
Compared with the apparent threshold voltage for KCNA10 in whole cell
recordings, the considerably more negative threshold observed in
single-channel recording may be due in large part to the higher
resolution of the latter. Fit of the single-channel I-V
plot shown in Fig. 9B indicates the KCNA10 single-channel conductance is ~11 pS (linear fit between
40 and +40 mV).
Oocytes that were not injected, injected with water, injected with
other clones, or injected and did not express hKCNA10 never exhibited single-channel currents like those shown in Fig. 9.


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Fig. 9.
Single-channel conductance of hKCNA10 is ~11 pS. A:
representative single hKCNA10 channel currents recorded from an
inside-out membrane patch excised from an oocyte that expressed 2.5 mA
of whole cell current at +40 mV. In this experiment, extracellular
(pipette) solution is Na-MES (2 mM K+), and cytoplasmic
(bath) solution is K-ND96 (96 mM K+). The command potential
is indicated next to each trace. Seal resistance
(Rseal) was 44 G . B: single-channel
I-V plot for hKCNA10. Slope of best-fit line yields a
unitary channel conductance of 11.3 pS. Reversal potential extrapolated
from this line is 96 mV, very close to calculated reversal
potential of K (EK) of 98.3 mV, indicating that
hKCNA10 is highly K selective.
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Conclusion
hKCNA10 is a voltage-gated channel highly selective for K with a
single-channel conductance of ~11 pS. Current is inhibited not only
by classical K channel inhibitors but also by specific blockers of CNG
cation channels. hKCNA10 belongs to a new class of K channel that
includes HERG, Eag ATK1, and BCNG-1 These channels have structural
features and pharmacological properties common to both voltage-gated
Shaker K channels and cyclic nucleotide-gated nonselective
cation channels.
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ACKNOWLEDGEMENTS |
G. V. Desir was supported by a Veteran Administration Merit Review
Award from the National Institute of Diabetes and Digestive and Kidney
Diseases (DK-48105B) and is an American Heart Association Established Investigator.
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FOOTNOTES |
*
R. Lang and G. Lee contributed equally to this work.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. V. Desir,
Dept. of Medicine, Sect. of Nephrology, Yale Univ. School of Medicine,
2073 LMP, 333 Cedar St., New Haven CT 06510 (E-mail
gary.desir{at}yale.edu).
Received 19 July 1999; accepted in final form 11 February 2000.
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REFERENCES |
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