Modulation of potassium channel gating by coexpression of
Kv2.1 with regulatory Kv5.1 or Kv6.1
-subunits
J. W.
Kramer,
M. A.
Post,
A. M.
Brown, and
G. E.
Kirsch
Department of Physiology and Biophysics, Rammelkamp Research Center,
MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio
44109
 |
ABSTRACT |
We have
determined the effects of coexpression of Kv2.1 with electrically
silent Kv5.1 or Kv6.1
-subunits in
Xenopus oocytes on channel gating.
Kv2.1/5.1 selectively accelerated the rate of inactivation at
intermediate potentials (
30 to 0 mV), without affecting the rate
at strong depolarization (0 to +40 mV), and markedly accelerated the
rate of cumulative inactivation evoked by high-frequency trains of
short pulses. Kv5.1 coexpression also slowed deactivation of Kv2.1. In
contrast, Kv6.1 was much less effective in speeding inactivation at
intermediate potentials, had a slowing effect on inactivation at strong
depolarizations, and had no effect on cumulative inactivation. Kv6.1,
however, had profound effects on activation, including a negative shift of the steady-state activation curve and marked slowing of deactivation tail currents. Support for the notion that the Kv5.1's effects stem
from coassembly of
-subunits into heteromeric channels was obtained
from biochemical evidence of protein-protein interaction and
single-channel measurements that showed heterogeneity in unitary conductance. Our results show that Kv5.1 and Kv6.1 function as regulatory
-subunits that when coassembled with Kv2.1 can modulate gating in a physiologically relevant manner.
rat brain; Xenopus oocytes; delayed rectifier; DRK1; IK8; K13
 |
INTRODUCTION |
VOLTAGE-GATED POTASSIUM channels are transmembrane
proteins consisting of four
-subunits arranged in radially symmetric
fashion around a central aqueous pore. In mammals, a large family of
genes encodes
-subunits: Kv1 (homologous to
Drosophila Shaker), Kv2 (Shab), Kv3
(Shaw), Kv4
(Shal), Kv5, Kv6, and Kv8.
Heterologous expression of homotetrameric channels of the Kv1-Kv4
subfamilies display distinctive gating characteristics associated with
variations in the primary sequence. In addition, gating is known to be
modified through assembly of heterotetramers consisting either of
different
-subunits (14) or
-subunits with accessory
-subunits
(26). Heteromeric assembly has been thought to be restricted to members of the same Kv subfamily (6, 21). However, recent evidence indicates
that heteromeric assembly with functional consequences can occur
between
-subunits from different subfamilies. The Kv2 (Shab) subfamily is particularly
noteworthy in this regard. Only two mammalian members have been
identified, Kv2.1 and Kv2.2, and both express channels with very
similar gating (10, 13). However, when Kv2.1 is coexpressed with
certain other
-subunits that, by themselves, do not produce
electrically functional channels, heteromeric channels with markedly
altered gating kinetics are formed (12, 25). Moreover, regulation of
gating by electrically silent
-subunits is not restricted to the Kv2
subfamily. Recent evidence indicates the existence of regulatory
-subunits that interact with members of the
Shal (16) and Kv3
(Shaw) (28) subfamilies as well.
We demonstrated previously that coexpression of Kv2.1 with the
electrically silent Kv6.1 (formerly known as K13; Ref. 9) results in
potassium channels that had slower kinetics of deactivation (25).
Evidence for heteromeric assembly was obtained using a yeast two-hybrid
assay that demonstrated interactions between the
NH2 termini of each
-subunit
and through a functional assay that showed altered sensitivity to a
pore-blocking drug and markedly slower closing rates upon
repolarization (deactivation). No change in inactivation kinetics was
reported. Recently, however, Kv8.1, Kv6.1 (4, 27, 28), and to a much
lesser extent Kv5.1 (Ref. 28; formerly known as IK8; Ref. 9) were shown
to slow the time course of Kv2.1 inactivation.
Kv2.1 channels show characteristically slow kinetics of inactivation
(5-20 s) that may be related to C-type inactivation (7) originally
observed in the Kv1 (Shaker) class
of channels. The physiological relevance of this type of inactivation
is unclear in view of its extremely slow time course. Its mechanism
likewise is uncertain but appears to involve pore-lining residues
rather than the cytoplasmic NH2
terminus that is responsible for N-type inactivation (5, 31). Moreover,
unlike fast, N-type inactivation in
Shaker, slow inactivation in Kv2.1
appears to involve a substantial contribution from a pathway leading
directly from closed to inactivated states of the channel (19). Several
studies (23, 24) have shown that all four
-subunits participate
cooperatively in C-type inactivation. Therefore, if each
-subunit is involved in the inactivation process, then it is
likely that the formation of heteromeric channels can influence the
kinetics of inactivation, as was observed in Kv2.1/Kv8.1 coexpression
(27). We previously obtained yeast two-hybrid evidence for interactions
between Kv2.1 and Kv5.1 but presented no data concerning a functional
interaction. Our goal in the present paper was to compare the effects
on channel gating of coexpression of Kv5.1 and Kv2.1 with those
obtained in Kv2.1/Kv6.1 coexpression. We paid particular attention to
regulatory effects of coexpression on the rates of onset and recovery
from inactivation over a wide range of voltages. We found that
coexpression of Kv5.1 or, to a lesser extent Kv6.1, with Kv2.1 markedly
accelerated closed channel inactivation such that both its voltage
range and time course became more appropriate for a potential role in
regulating membrane excitability.
 |
METHODS |
Yeast two-hybrid system and fusion protein constructs.
The MATCHMAKER Yeast Two-Hybrid System (Clontech Laboratories, Palo
Alto, CA) was used to assay for protein-protein interactions. The
NH2 termini of the voltage-gated
potassium channels Kv1.2 nt 1-492 (amino acids 1-164), Kv2.1
nt 1-504 (amino acids 1-169), Kv5.1 nt 1-528 (amino
acids 1-176), and Kv6.1 nt 1-627 (amino acids 1-209)
were cloned into yeast two-hybrid vectors pGAD424 (LEU2,
ampr) and pGBT9 (TRP1,
ampr). The Kv channel inserts
were generated by PCR using upstream oligonucleotides with an
EcoR I site and downstream
oligonucleotides with a stop codon/Sal
I site to allow directional cloning and correct termination of the
fusion protein. All sequences were verified by manual sequencing. Yeast
strain Y190 (MATa, Ura3-52, His3-200,
Lys2-801, Ade2-101, Trp1-901, Leu2-3,
112, gal4
, gal80
, cyhr2,
LYS2::GAL1UAS-HIS3TATA-HIS3,
URA3::GAL1UAS-GAL1TATA-lacZ)
(Clontech Laboratories, Palo Alto, CA) was used for all experiments.
Y190 cells were transformed with the plasmid constructs of interest (0.1-1 µg of each) and plated on
Trp/
Leu media to
select for cells harboring both vectors.
LacZ expression assays were performed by lifting Y190 colonies onto grade 410 filter paper (VWR, West Chester, PA), freeze thawing in liquid nitrogen, and incubating at
37°C for up to 6 h on filter paper soaked in Z buffer (in mM: 60 Na2HPO4,
40 NaH2PO4,
10 KCl, and 1 MgSO4, pH 7.0) to
which (final concentrations) 0.27%
-mercaptoethanol and 0.33 mg/ml 5-bromo-4-chloro-3-indoyl-
-D-galactopyranoside
(X-gal) (Research Biochemicals, Natick, MA) had been added. All
reagents used, unless otherwise stated, were purchased from Sigma
Chemical (St. Louis, MO), Fisher Scientific (Pittsburgh, PA), or
Boehringer Mannheim (Indianapolis, IN).
RNA synthesis.
Kv2.1, Kv5.1, and Kv6.1 in Bluescript
SK
were linearized at the
3' Not I site, proteinase K
treated, phenol chloroform extracted, precipitated, and resuspended in
water at a concentration of ~1 µg/µl. Linearized cDNA was
transcribed in vitro with the mMessage mMachine kit (Ambion, Austin,
TX) according to the manufacturer's directions. The final cRNA
products were suspended in 100 mM KCl, and their concentration and
integrity were checked by formaldehyde agarose gel electrophoresis.
Oocyte preparation.
Stage V-VI Xenopus oocytes were
defolliculated by collagenase treatment (2 mg/ml for 1.5 h) in a
Ca-free buffer solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM
MgCl2, 5 mM HEPES, and 100 µg/ml
gentamicin, pH 7.6). The defolliculated oocytes were injected with 46 nl cRNA solution (in 100 mM KCl) and incubated at 19°C in culture
medium (100 mM NaCl, 2 mM KCl, 1.8 mM
CaCl2, 1 mM
MgCl2, 5 mM HEPES, 2.5 mM pyruvic
acid, and 100 µg/µl gentamycin, pH 7.6). Oocytes were injected with
2.5 ng/µl Kv2.1 cRNA for all experiments. In coexpression
experiments, 25 ng/µl Kv5.1 or Kv6.1 was coinjected with 2.5 ng/µl
Kv2.1.
Whole cell current recording.
Standard two-microelectrode voltage-clamp techniques were used to
measure macroscopic current (8). Micropipette tip resistance was
0.5-1 M
when filled with 3 M KCl plus 1% agar. The bath
solution was a normal Ringer solution of the following composition (in mM): 120 NaCl, 2.5 KCl, 2 CaCl2,
and 10 HEPES, pH 7.4. In some experiments, 120 mM NaCl was replaced
with KCl (K-Ringer solution). Recordings were performed 2-6 days
after cRNA injection. Voltage commands and data acquisition were
controlled with pCLAMP software (Axon Instruments, Foster City, CA).
All measurements were taken at room temperature (21-23°C), and
data were filtered at 1 kHz and digitized at 5 kHz. The current traces
were corrected for leak off-line except where noted. Data analysis and
curve fitting were performed using pCLAMP software. The choice of
biexponential over monoexponential functions to fit kinetic data was
based on an F ratio test
(P < 0.05) that takes into account
the increased number of free parameters. Where appropriate, data are
expressed as means ± SE. Statistical significance of the difference
between means was evaluated using a two-tailed Student's
t-test
(P < 0.05 or 0.01, as noted).
Single-channel recording.
Cell-attached patch recording was performed after manual removal of the
vitelline envelope. Isotonic KCl bathing solution was used to zero the
resting potential, and the absence of resting membrane potential was
verified by rupturing the membrane patch at the end of each experiment
to allow direct intracellular potential measurement. Holding and test
potentials applied to the membrane patch during the experiment are
reported as conventional intracellular potentials. Channels were
activated by rectangular test pulses from negative holding potentials.
Current records were low pass filtered at 1-2 kHz (
3 dB,
4-pole Bessel filter) then digitized at 5-10 kHz. Linear leakage
and capacitative currents were subtracted digitally using the smoothed
average of 10-20 null traces in which no channel openings could be
detected. Openings were identified using a half-amplitude criterion
(Transit analysis program, Ref. 30). Amplitude histograms of the
idealized records were fit to Gaussian distributions using a maximum
likelihood estimate. Events distributions of <0.3 ms duration were
excluded from fitting to avoid error introduced by the limited
recording bandwidth (1 kHz).
 |
RESULTS |
Coexpression of Kv2.1 with either Kv5.1 or Kv6.1 slows deactivation.
We previously showed that coexpression of Kv2.1 and Kv6.1 resulted in
currents that deactivated extremely slowly upon repolarization (25).
Qualitatively similar results were obtained for coexpression of Kv2.1
and Kv5.1. Figure 1 shows typical records
obtained from oocytes that expressed either Kv2.1
(A) or Kv2.1/5.1
(B). Currents were evoked by a
two-step protocol consisting of a conditioning step to +60 mV that
provided maximum activation, followed by a return to various
repolarizing test steps (
100,
80,
60, and
40 mV steps are illustrated). This pulse protocol evoked inward tail currents that subsided with an exponential time course. In both
sets of recordings, tail current time course was accelerated by more
negative return potentials; however, at test potentials more positive
than
80 mV, coexpression of Kv2.1/5.1 (Fig.
1B) resulted in tail currents that
decayed more slowly than Kv2.1 (Fig.
1A). Figure
1C shows that the time constant of
deactivation for both Kv2.1 (solid circles) and Kv2.1/5.1 (open
squares) decreased monotonically with increasingly negative test
potentials and that the two curves merged at potentials more negative
than
80 mV. At more positive potentials, the curves diverged
such that Kv2.1/5.1 time constants were much slower than those of
Kv2.1. Our results indicate that heteromer assembly slowed the
deactivation process at intermediate but not at extremely negative
potentials. This effect was different from that observed previously in
Kv2.1/6.1 coexpression (open triangles) in that the magnitude of the
Kv2.1/5.1 effect was much smaller and fast deactivation could be
restored at sufficiently negative voltage steps.

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Fig. 1.
Effect of Kv5.1 on deactivation. Channels were activated by
conditioning pulses to +60 mV that evoked outward current. Repolarizing
test pulses of 100 to 20 mV ( 100, 80,
60, and 40 are illustrated in
A and
B) evoked inward tail currents
(Im) in oocytes
bathed in K-Ringer solution. Linear leakage and capacitative
currents were corrected on-line using a P/4 subtraction
routine. A: typical records
obtained from oocytes injected with Kv2.1 alone.
B: records obtained from oocytes
coinjected with Kv2.1 and a 10-fold excess of Kv5.1. Decay of tail
currents was fit to monoexponential functions, and time constant was
plotted as a function of test pulse potential
(Em;
C). Values are means ± SE
(n = 5).
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Coexpression potentiates inactivation.
During maintained depolarization, channels close by the process of
inactivation. Thus, in an oocyte expressing Kv2.1 alone, a test pulse
to +40 mV for 15 s (Fig.
2A)
evoked a slow, monoexponentially decaying current with a time constant
(
) of 7 s. Coexpression of Kv2.1 and Kv5.1
-subunits did not
alter the onset of inactivation significantly (
= 7 s); however,
coexpression of Kv2.1 and Kv6.1 resulted in a much slower onset of
inactivation (
= 32 s) compared with either Kv2.1 alone or
Kv2.1/5.1. The onset of inactivation was relatively insensitive to test
pulse potential in the range 0 to +40 mV (Fig.
2B) whether Kv2.1 was expressed
alone or with either Kv5.1 or Kv6.1. Thus, throughout the voltage range
in which channels were strongly activated, the time constants for
inactivation were either not significantly affected (Kv2.1/5.1
coexpression) or were markedly increased (Kv2.1/Kv6.1). On this basis,
we might be tempted to suggest that coexpression of regulatory subunits with Kv2.1 has, at most, an inhibitory effect on inactivation.

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Fig. 2.
Effects of regulatory subunits on development of inactivation at
strongly depolarized test potentials.
A: test pulse potentials of +40 mV
from a holding potential of 80 mV evoked outward current that
decayed exponentially. B: time
constants obtained from monoexponential fits of decay of current during
long test pulses were plotted on a logarithmic scale vs. test pulse
potential for Kv2.1, coexpressed with Kv5.1 or with Kv6.1
(n = 5-8 cells/group).
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A very different picture emerged when we investigated inactivation at
potentials closer to the foot of the activation range (see Fig.
4B). These experiments used a
double-pulse protocol (Fig.
3A,
inset) consisting of a depolarizing
prepulse of variable duration to initiate inactivation (amplitude,
30 to 0 mV), followed immediately by a +60-mV test pulse that
was sufficient to activate all available channels. As shown in Fig.
3A, peak test pulse currents decreased
with increasing prepulse duration, such that the envelope of the peaks
tracked the time course of inactivation. Peak current was plotted
against prepulse length, and data were fit to either monoexponential
(Kv2.1) or biexponential (Kv2.1/5.1 and Kv2.1/6.1) decay functions
(Fig. 3B). At a prepulse potential
of
30 mV, inactivation of Kv2.1 (Fig.
3B, solid circles) was quite slow with
a
of ~24 s. However, the onset of inactivation was accelerated ~100-fold when Kv2.1 was coexpressed with Kv5.1 (Fig.
3B, open squares) and ~10-fold by
Kv6.1 (Fig. 3B, open triangles). Over the range of potentials from
30 to 0 mV, the
of inactivation of Kv2.1 was characteristically voltage dependent (Fig.
3C, semilogarithmic plot) such that it
became progressively shorter with more positive prepulse potentials. In
contrast, for both Kv2.1/5.1 and Kv2.1/6.1 coexpression (Fig. 3,
C and
D), two time constants were required for an accurate fit, and their voltage dependence was reduced. In
Kv2.1/5.1, both fast and slow components of inactivation were significantly faster than the single component of Kv2.1. However, in
Kv2.1/6.1, although the fast component was significantly faster than
Kv2.1 (Fig. 3C), the slow component
(Fig. 3D) was either slower (test
potentials,
10 and 0 mV), unchanged (test potential
20
mV), or slightly faster (test potential
30 mV) than Kv2.1 alone.

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Fig. 3.
Regulatory subunits accelerate inactivation at low depolarization.
A: typical records obtained from an
oocyte expressing Kv2.1 alone using stimulus protocol shown in
inset; a variable-length,
constant-amplitude ( 30 mV is illustrated) conditioning pulse was
immediately followed by a short +60-mV test pulse to assess amount of
inactivation that developed during conditioning prepulse. Holding
potential was 80 mV. B: peak
test pulse current vs. prepulse duration for Kv2.1, Kv2.1/6.1, and
Kv2.1/5.1 at a prepulse amplitude of 30 mV. Smooth curves are
monoexponential (Kv2.1) or biexponential (Kv2.1/5.1 and Kv2.1/6.1) fits
to data. C: fast time constant
component vs. prepulse amplitude Kv2.1, Kv2.1/5.1, and Kv2.1/6.1 at
prepulse potentials of 0, 10, 20, and 30 mV
plotted semilogarithmically. Values are means ± SE
(n = 4 or 5). All time constants for
Kv2.1/5.1 and Kv2.1/6.1 are significantly different from Kv2.1
(P < 0.01).
D: slow time constant component vs.
prepulse amplitude. Because of its monophasic decay, Kv2.1 data are
same in both C and
D. Compared with Kv2.1, time constants
obtained in Kv5.1 and Kv6.1 coexpression experiments were either
significantly different at P < 0.01 (**) or P < 0.05 (*) levels or not
significantly different (ns). Each component in Kv5.1 and Kv6.1 data
had a relative amplitude of at least 0.2.
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The kinetic results obtained at the two different voltage ranges
suggested that transitions into the inactivated state from weakly
activated, closed states were markedly potentiated in Kv5.1/2.1 and, to
a lesser degree, in Kv6.1/2.1 coexpression. Moreover, the voltage
dependence of inactivation appeared to be altered by coexpression. To
test these ideas further, steady-state inactivation curves were
obtained using a two-pulse protocol consisting of a fixed-duration (40 s) prepulse that ranged from
120 to +20 mV in amplitude. After
the prepulse, a +40-mV test potential was applied to assess the
fraction of noninactivated channels. Figure 4A plots
normalized test pulse current vs. prepulse voltage. The data were fit
to Boltzmann functions (smooth curves). Coexpression of Kv2.1 with
either Kv5.1 or Kv6.1 caused a negative shift in the voltage dependence
of the steady-state inactivation curves as compared with Kv2.1.
Inactivation in Kv2.1/5.1 showed a half-maximal potential
(V0.5) of
57 mV, while Kv2.1/6.1 had a
V0.5 of
66 mV. These values differed considerably from Kv2.1, which had a V0.5 of
30
mV. The inactivation curves also differed in steepness of slope.
Coexpression of Kv2.1/5.1 and, to a lesser extent, Kv2.1/6.1 tended to
reduce the steepness of the curves. (Slope factors of 4.8, 6.4, and
11.1 mV, respectively, were required to fit data from Kv2.1, Kv2.1/6.1,
and Kv2.1/5.1.)

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Fig. 4.
Regulatory subunits cause a negative shift of voltage range for
inactivation. A: steady-state
inactivation curves obtained with conditioning potentials of 120
mV to +10 mV for 40 s and test potential to +40 mV. Peak currents
(normalized to maximum observed current in each cell) for Kv2.1,
Kv2.1/5.1, and Kv2.1/6.1 are plotted vs. conditioning membrane
potential. Values are means ± SE
(n = 4 or 5). Smooth curves are fits
to a Boltzmann function: h0 + (1 h0)/{1 + exp[(V V0.5)/k]},
where h0 is nonzero foot,
V is conditioning potential,
V0.5 is midpoint
potential, and k is a slope factor.
Fitted parameters for Kv2.1 were
V0.5 = 30.1 mV and k = 4.8 mV.
Values for Kv2.1/5.1 were
V0.5 = 56.6 mV and k = 11.1 mV.
Values for Kv2.1/6.1 were
V0.5 = 65.9 mV and k = 6.4.
B: steady-state activation curves were
obtained from current-voltage families using a series of 500-ms test
pulses from 70 to +80 mV from a holding potential of 80
mV. Normalized conductance was estimated from isochronal (2 ms) tail
currents at 80 mV. These experiments were performed in K-Ringer
solution. Values are means ± SE (n = 4-6). Smooth curve represents Boltzmann fits to data as
discussed in text. Fitted parameters for Kv2.1 were
V0.5 = 1.7
mV and k = 9.6 mV. Values for
Kv2.1/5.1 were
V0.5 = 18.5 mV
and k = 17.3 mV. Values for Kv2.1/6.1
were V0.5 = 9.4 mV and k = 11.8 mV.
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Shifts in the steady-state inactivation curves along the voltage axis
(Fig. 4A) could not be predicted
from the shifts of the activation curves because as shown in Fig.
4B, the voltage range for Kv2.1/5.1
activation was shifted in a direction opposite to that of the
inactivation curve (Fig. 4A). In
Kv2.1/6.1, both curves were shifted in the same direction, but
inactivation (
35 mV shift) was affected to a much greater degree
than activation (
8 mV shift). These data indicate that a major
effect of coexpression was to shift the range of potentials in which
inactivation occurred to a much more negative region relative to that
of activation, consistent with the hypothesis that coexpression
promotes inactivation from partially activated closed states of the
heteromeric channels.
Coexpression speeds recovery from inactivation.
Because coexpression of regulatory subunits had a marked effect on the
development of inactivation, we determined whether recovery from
inactivation also was affected. Recovery was measured using a
four-pulse protocol (Fig.
5A) that
consisted of a brief initial prepulse
(P1) to assess the maximum
available current, followed by a conditioning pulse to 0 mV for
10-15 s to elicit a substantial amount of inactivation.
Inactivated channels were allowed to recover for a varying length of
time at voltages ranging from
120 to
60 mV before
application of a test pulse (P3)
to +40 mV. The fractional recovery from inactivation was determined as
IP3/IP1.
Figure 5B plots the time course of
recovery at
90 mV. Points were fit with a monoexponential
function to obtain time constants of recovery. Coexpression
of Kv2.1 with either Kv5.1 or Kv6.1 accelerated the time course of
recovery from inactivation at
90 mV. Average recovery
values
were 0.8 and 0.3 s for Kv2.1/6.1 and Kv2.1/5.1, respectively, compared
with 1.6 s for Kv2.1. Figure 5C shows
values of recovery over a wide range of potentials. Recovery from
inactivation for Kv2.1/5.1 was consistently faster than Kv2.1
throughout the range tested (
120 to
60 mV,
= 0.2-0.8 s). Over the range of potentials tested, Kv2.1 and
Kv2.1/6.1 showed a 20-fold change in the time constant as compared with
a 4-fold change for Kv2.1/5.1.

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Fig. 5.
Effect of regulatory subunits on recovery from inactivation.
A: a test pulse to +40 mV
(P1,
inset) for 0.25 s (to determine
maximum current) was followed by a conditioning pulse
(P2) to 0 mV (Kv2.1 and
Kv2.1/5.1) or 10 mV (Kv2.1/6.1). After a variable duration (10 ms to 25 s) recovery interval at a constant amplitude of 120 to
60 mV, another test pulse
(P3) to +40 mV assessed amount
of recovery. B:
P2 duration at 90 mV vs.
amount of recovery
(IP3/IP1).
Data were fit with a single exponential equation to obtain time
constants of recovery. C: time
constants vs. P2 amplitude,
plotted semilogarithmically. Values are means ± SE
(n = 4-8).
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Kv5.1 facilitates cumulative inactivation.
Potassium channels often show cumulative inactivation (1), defined as a
progressive decline in the amplitude of test pulse currents that occurs
during a train of repetitive depolarizing test pulses, each of which is
too brief to allow observable inactivation during the pulse. Therefore,
we compared the time course of cumulative inactivation in Kv2.1, with
and without expression of regulatory subunits, under conditions that
would be appropriate for repetitive firing in excitable cells. Figure
6A shows
that for Kv2.1/5.1 coexpression, a rapid decline in the amplitude of
successive test pulse currents was observed during a stimulus train
consisting of short (40 ms) depolarizations to +40 mV, each pulse
separated by a 20-ms period at the holding potential of
80 mV
(Fig. 6A, inset). Under the same conditions,
Kv2.1 and Kv2.1/6.1 showed much slower cumulative inactivation (Fig.
6B). Plots of normalized test pulse
current vs. time were accurately fit to monoexponential decay functions
with
values of 1.6 and 1.2 s, respectively, for Kv2.1 and
Kv2.1/6.1. Cumulative inactivation of Kv2.1/5.1, in contrast, followed
a biexponential decay with fast and slow
values, respectively, of
0.1 and 1.7 s. Continuous depolarization at +40 mV, by comparison,
yielded inactivation
values of 7 s in Kv2.1 and Kv2.1/5.1 and 32 s
in Kv2.1/6.1 (Fig. 2A). Therefore, in all cases, inactivation that proceeds from preopen closed states, which are populated during the rising phase of the test pulse current
(note that a 40-ms test pulse achieved <85% of full activation), was
more rapid than that which proceeds from open channels. Moreover, Kv2.1/5.1 coexpression was particularly effective in promoting this
form of inactivation.

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Fig. 6.
Coexpression of Kv2.1/5.1 accelerates cumulative inactivation.
A: typical current traces obtained
from Kv2.1/5.1 by repetitive pulsing
(inset). Currents evoked by first 10 pulses in train are superimposed; successive test pulses produced
progressively smaller amplitude currents until a steady state was
reached after pulse 4. For clarity,
capacitative transient at beginning of recording (time = 0-1.8 ms)
has been blanked. B: peak current vs.
elapsed time from start of pulse train. Currents were normalized to
initial test pulse current amplitude. Data points are means ± SE
(n = 5). Smooth curves show
monoexponential [Kv2.1 and Kv2.1/6.1, time constant ( ) = 2 s] or biexponential fits (Kv2.1/5.1,
fast = 0.1 s, relative
amplitude 0.5; and slow = 1.7 s, relative amplitude 0.2).
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Kv5.1 and Kv6.1 NH2 termini interact
specifically with Kv2.1 NH2 terminus.
Yeast two-hybrid assays allow detection of protein-protein interactions
by bringing a transcription factor's DNA binding and activation
domains together, allowing the subsequent transcription of a reporter
gene. The two parts of the transcription factor are brought together by
creating fusion proteins with, in this case, portions of Kv channels
that may interact. To test for these interactions, X-gal assays were
performed on Y190 cells harboring combinations of the activation domain
and DNA binding domain in fusion with Kv2.1, Kv5.1, and Kv6.1 as well
as the parental pGAD424 and pGBT9 plasmids (Table
1). Interaction strength was determined qualitatively from the intensity and speed of color development. Regardless of the vector background (i.e., DNA binding domain or
activation domain), Kv2.1 strongly interacted with itself, Kv5.1, and
Kv6.1 (indicated by ++). None of the Kv channel constructs alone was
positive (no color development, indicated by
), nor did Kv6.1
interact with itself. In addition, Kv1.2 failed to interact with any of
the Kv channels tested except itself. The
NH2 termini of Kv5.1, however, did
show a slight self-interaction (indicated by
/+). These results
confirm our previous report (25) and extend our observations to include
interactions between Kv2.1 and Kv5.1.
View this table:
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|
Table 1.
X-gal assay of Y190 yeast cells harboring activation domain and DNA
binding domain fusion protein constructs of K channel NH2
termini
|
|
Heteromeric assembly of Kv2.1/5.1 channels.
We previously demonstrated the participation of Kv6.1 in the assembly
of Kv2.1/6.1 heteromers based on evidence that coinjection of the two
-subunits results in channels with markedly altered pore properties
(25). We showed that Kv2.1/6.1 was resistant to block by
tetraethylammonium (TEA), presumably because Kv6.1 contains a valine
residue in place of the tyrosine, present in both Kv2.1 and Kv5.1, that
is required for high sensitivity to block by TEA (11). As predicted by
the presence of the critical tyrosine in Kv5.1, no difference in TEA
block was observed between Kv2.1 and Kv2.1/Kv5.1 (data not shown).
However, single-channel conductance also is regulated by the
interaction of all four subunits that make up the pore in
heterotetrameric channels (17). Therefore, we measured single-channel
conductance as an assay for changes in pore properties that might be
attributable to heteromeric Kv2.1/Kv5.1 channel formation. Figure
7,
A-C,
shows typical single-channel records obtained from cell-attached
patches exposed externally to 120 mM KCl solution to maximize the
conductance (18). Under these conditions (Fig.
7D), Kv2.1 channels had a
conductance of 14.8 pS, whereas in Kv2.1/5.1-injected oocytes, we
observed two classes of channels: 12.4 pS (all 13 patches) and 4.2 pS
(6 of 13 patches). The differences between the 14.8- and 12.4-pS as well as the 12.4- and 4.2-pS conductance levels were statistically significant (P < 0.04). Therefore,
we interpret the 12.4-pS channels as a heteromer of Kv2.1 + Kv5.1, and
the 4.2-pS conductance suggests the possibility of multiple classes of
heteromeric channels arising from different mixtures of subunits. It is
noteworthy that the multiple time constants obtained from analysis of
macroscopic inactivation experiments (Fig. 3,
C and
D) also support the idea of channel
heterogeneity.

View larger version (20K):
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|
Fig. 7.
Effect of Kv5.1 on single-channel conductance.
A-C:
typical records obtained in cell-attached patches exposed externally to
120 mM KCl solution, in oocytes expressing Kv2.1 alone
(A) or Kv2.1 + Kv5.1
(B and
C). Data are pooled from 7 Kv2.1
patches and 13 Kv2.1 + Kv5.1 patches that were injected with
an RNA ratio of 1 Kv2.1:10 Kv5.1 to increase probability of observing
heterotetramers. Test pulses to +80 mV evoked single-channel openings
of 1.2 pA in Kv2.1 (A) but only 1.0 pA in Kv2.1/5.1 (B). We observed 2 classes of channels in all patches from Kv2.1 + Kv5.1-injected oocytes:
12.4 pS (all 13 patches) and 4.2 pS (6 of 13 patches). Presence of
multiple classes in coexpression is consistent with different
combinations of subunits during channel assembly. Kv2.1 channels
averaged 14.8 pS (n = 7). Records were
obtained from a holding potential of 80 mV. Data were low-pass
filtered at 1 kHz. D: single-channel
current-voltage plots from 3 classes of channels. Values are means ± SE (n = 7, 13, and 6, respectively, for 15-, 12-, and 5-pS channels). Data are fit by linear
regression to obtain single-channel conductances. Difference between
14.8- and 12.4-pS conductance levels was statistically significant
(P < 0.05).
|
|
 |
DISCUSSION |
Modulation of gating by regulatory
-subunits.
We have presented here the first evidence of heteromeric assembly of
Kv2.1/5.1 channels and the first detailed kinetic observations of the
effects of heteromultimerization of Kv2.1
-subunits with electrically silent Kv5.1 and Kv6.1
-subunits. The two subunits can
be distinguished on the basis of how they affect the rate of channel
closure via two gating reactions: deactivation upon repolarization and
inactivation during maintained depolarization (Table
2). Our results show clearly that Kv5.1
accelerates inactivation but has relatively little effect on
deactivation. Kv6.1 accelerates inactivation less effectively but has a
pronounced slowing effect on deactivation. A separate question is
whether the effects on inactivation are associated with pathways
involving either closed or open states of the channels. Klemic et al.
(19) proposed that in Kv2.1 channels transitions from preopen closed
states to the inactivated state are much faster than those from open to
the inactivated states. Our observations support this idea by showing
that coexpression of Kv2.1 with Kv5.1 markedly accelerated inactivation
at intermediate potentials near the foot of the activation curve
(
30 to
10 mV) but had almost no effect on the rate of inactivation at strong depolarizations approaching the plateau region
(>40 mV). Moreover, coexpression of Kv2.1 with Kv6.1 caused a marked
slowing of inactivation at strong depolarizations (Fig. 2A) but acceleration at intermediate
potentials (Fig. 3B). These results
indicate that, for Kv2.1, two pathways for inactivation exist and that
they can be regulated independently of one another.
Two lines of evidence support the notion that these kinetic effects are
the result of coassembly of Kv2.1 and Kv5.1 or 6.1 subunits into a
functional channel rather than a regulatory effect of Kv5.1 or Kv6.1 on
the gating properties of homotetrameric Kv2.1 channels, such as that of
-subunits on voltage-gated potassium or sodium channels, (26, 15).
First, the yeast two-hybrid assay provided evidence for protein-protein
interactions between the NH2
termini of these subunits. It is noteworthy that these interactions
showed a high degree of specificity; only interactions between Kv2.1
and Kv5.1 or Kv6.1 were observed. Second, single-channel recordings in
oocytes coinjected with Kv2.1 and Kv5.1 provided evidence for channels
with unitary conductance distinct from that observed in oocytes
injected with Kv2.1 alone. This observation indicates a change in pore
structure that can most readily be explained by heterotetramer
formation.
Electrophysiological analysis of Kv2.1/5.1 coexpression revealed
currents with unique inactivation kinetics; the onset of inactivation
at strongly depolarized potentials was unchanged, whereas a marked
acceleration was observed at potentials near the foot of the activation
curve. These results indicated that inactivation from partially
activated channels was potentiated. This was further substantiated by a
marked shift of the steady-state inactivation curve in the
hyperpolarized direction. A potentially important consequence of the
regulatory influence of Kv5.1 was revealed in a comparison of the time
course of cumulative inactivation during repetitive stimulation, which
showed that Kv2.1/5.1 inactivated much more rapidly than either
Kv2.1/6.1 or Kv2.1 alone. However, the kinetics of recovery from
inactivation also were much faster in Kv2.1/5.1 than in Kv2.1. Because
both entry into and recovery from inactivation were accelerated, the
balance between these two opposing tendencies would be expected to
govern the relationship between the rate of cumulative inactivation and
stimulus frequency. At low stimulus frequencies where Kv2.1/5.1
channels have a greater chance to recover from inactivation, the rate
of cumulative inactivation would be expected to be slower as compared
with high stimulus frequency, where inactivation would be expected to
dominate over recovery. Thus the kinetic effect of Kv5.1 on Kv2.1/5.1
heteromers appeared to be directed primarily toward acceleration of the
on and off rates of inactivation and a negative shift of its
steady-state voltage dependence. This result is in marked contrast to a
recently published study (28) that concluded, based on results obtained only with strong depolarizing pulses, that the primary regulatory influence of Kv5.1 (and Kv6.1) is to slow the kinetics of inactivation. In addition, Kv2.1/5.1 heteromers also displayed altered activation kinetics that would tend to hold channels open at physiologically relevant membrane potentials (
80 to
20 mV). Taken
together, the modulatory effects associated with Kv2.1/5.1 heteromers
would allow greater flexibility in the gating of delayed rectifier
channels over a voltage range that is critical for control of membrane excitability.
In contrast, inactivation may be secondary to activation gating as the
regulatory target for Kv6.1 in coexpression with Kv2.1. We base this on
our previous finding (25) that deactivation was slowed by a factor of
5- to 15-fold over the range
60 to
100 mV and the
steady-state voltage dependence of activation was shifted toward more
negative potentials. Both of these effects would tend to increase
potassium conductance over a physiologically significant potential
range corresponding to the voltage region encompassing the action
potential threshold and the peak of the hyperpolarizing afterpotential.
The effects on inactivation, although qualitatively similar to those of
Kv5.1, were much smaller in magnitude and, as a result, had little
effect on cumulative inactivation.
Recent studies indicate that, like Kv5.1 and 6.1, the electrically
silent Kv8.1 subunit also functions as a regulatory
-subunit of
Kv2.1 (12). Although initial reports stressed an inhibitory effect of
Kv8.1 on Kv2.1 expression (12), more recent studies (4, 27) suggest
modulation of channel gating as its primary function. The effects of
Kv8.1 on Kv2.1 (or Kv2.2) gating most closely resemble those of Kv5.1
(Table 2). Thus Kv8.1 shifts the voltage dependence of inactivation by
approximately
40 mV, whereas activation is shifted by less than
+10 mV (4, 27). Like Kv5.1, Kv8.1 produces a relatively small slowing
of deactivation (4). However, unlike Kv5.1, Kv8.1 produces a marked
slowing of inactivation during strong depolarizing test pulses (27). In
this regard, the Kv8.1 kinetic effect more closely resembles that of
Kv6.1. It should be noted, however, that Kv6.1's inhibitory effect on
inactivation at strongly depolarized potentials was reversed at
intermediate test potentials. Whether Kv8.1 has similar characteristics
has not been determined.
Implications for inactivation gating.
The mechanism of slow inactivation in potassium channels is not
thoroughly understood. Our results, however, place important constraints on gating models. Inactivation of delayed rectifier potassium current in molluscan neurons has been described as a voltage-
and state-dependent process where the transition to the inactivated
state from closed states is more favorable than from the open state
(1). Similarly, for Kv2.1 expressed in oocytes, slow inactivation has
been modeled as a state-dependent process that is most accessible from
a nonconducting, preopen state (19). Our results indicate that, in
Kv2.1, the inactivation process for fully activated open channels is
regulated differently from that which occurs in weakly activated closed
channels. For instance, Kv2.1/6.1 coexpression results in channels with
markedly slower inactivation during strong depolarization but faster
inactivation at intermediate potentials. Similarly, in Kv2.1/5.1
coexpression, the time course of inactivation during strong
depolarization was not affected, whereas inactivation at potentials
near activation threshold was accelerated 100-fold. These results are
consistent with the notion that inactivation is highly state dependent
such that inactivation from channels that are predominantly in the open
state can be much slower than inactivation from channels in preopen
closed states (19). Thus, regardless of the effect of Kv5.1 or Kv6.1
subunits on the time constant of inactivation during long-lasting,
strong depolarizing pulses, the effect on inactivation kinetics at
negative test potentials was always one of facilitation, as evidenced
by a negative shift in the steady-state voltage dependence of
inactivation and faster time constants for both onset and recovery. A
second implication concerning mechanisms of inactivation relates to the
idea of coupling between the inactivation and activation processes. In
voltage-gated sodium channels, the two processes are coupled such that
inactivation derives its voltage dependence through a tight coupling to
activation (2). Tight coupling also can be detected in delayed recovery
imposed by the necessity for channels to deactivate before recovery can
proceed upon repolarization (20). In contrast, our results suggest a much looser coupling in Kv2.1. Thus coexpression of Kv2.1/5.1 gave a
+15-mV shift in the voltage dependence of activation that was
accompanied by a
30-mV shift of the inactivation curve.
Similarly, for Kv2.1/6.1, the deactivation process was slowed by a
factor of 10, whereas the recovery from inactivation showed either no change or a modest acceleration. These results together with the observation of a "foot" or "U-shaped" configuration of the
steady-state inactivation curve (19) suggest that in Kv2.1 recovery
from inactivation can take place from open channels and that closed channels can inactivate.
Potential physiological significance of
-subunit
regulation.
Functional diversity achieved through assembly of heteromeric channels
from phenotypically distinct
-subunits is a hallmark of the Kv1 and
Kv3 subfamilies. In contrast, the Kv2 subfamily has only two
phenotypically indistinguishable mammalian members (Kv2.1 and Kv2.2).
Our results suggest that in this subfamily functional diversity is
promoted through heteromeric channel assembly with Kv5.1 and Kv6.1
-subunits. Moreover, the regulatory influence of these subunits is
clearly distinguishable: Kv5.1 primarily accelerates inactivation,
whereas Kv6.1 primarily slows deactivation. The potential physiological
consequences can be inferred from measurement of cumulative
inactivation during repetitive pulsing. Under conditions that simulate
high-frequency trains of brief impulses, we found that Kv2.1/5.1 but
not Kv2.1/6.1 showed a marked acceleration of the rate of cumulative
inactivation. Slow recovery from inactivation has also been suggested
as a factor in promoting cumulative inactivation (3). However, under
our stimulus regimen (40-ms depolarizing pulses, 20-ms interpulse
intervals), changes in recovery rates cannot play a role because no
significant recovery occurred during the brief interpulse interval.
Under these experimental conditions, the rapidity of inactivation
during the rising phase of the test pulse currents, rather than
differences in recovery during the interpulse interval, was the major
determinant. Thus the much greater acceleration of inactivation in
Kv2.1/5.1 than in Kv2.1/6.1 may account for the effect on cumulative
inactivation. Cumulative inactivation in neurons is thought to be an
important determinant patterning repetitive spike discharges (29) and in frequency-dependent spike broadening duration of trains of repetitive pulses (22). Our results suggest that a switch from either
predominantly Kv2.1 homotetramers or Kv2.1/6.1 heterotetramers to more
rapidly inactivating Kv2.1/5.1 heterotetramers would increase the
excitability of neurons during repetitive firing by increasing spike
frequency and prolonging the action potential. On the other hand,
compared with Kv2.1 homotetramers, Kv2.1/6.1 heterotetramers require a
much longer time to close upon repolarization and, therefore, might be
expected to prolong the refractory period that determines the
interspike interval and thereby reduce spike frequency during repetitive firing.
 |
ACKNOWLEDGEMENTS |
We thank W.-Q. Dong and C.-D. Zuo for expert oocyte injection and
culture.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
NS-29473 (to G. E. Kirsch), NS-23877, HL-36930, and HL-55404 (to A. M. Brown); National Research Service Award 93849 (to M. A. Post); and a
grant-in-aid from the American Heart Association, Northeast Ohio
Affiliate (to J. W. Kramer).
Address for reprint requests: G. E. Kirsch, MetroHealth Medical Center,
Rammelkamp Bldg. R327, 2500 MetroHealth Dr., Cleveland, OH 44109.
Received 22 October 1997; accepted in final form 19 February 1998.
 |
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