1Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, Health Sciences Centre, Calgary, Alberta T2N 4N1; and 2Department of Biological Sciences, The University of Alberta, Edmonton, Alberta T6G 2E9 and Bamfield Marine Station, Bamfield, British Columbia V0R 1B0, Canada
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ABSTRACT |
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Grigoriev, Nikita G., J. David Spafford, and Andrew N. Spencer. Modulation of Jellyfish Potassium Channels by External Potassium Ions. J. Neurophysiol. 82: 1728-1739, 1999. The amplitude of an A-like potassium current (IKfast) in identified cultured motor neurons isolated from the jellyfish Polyorchis penicillatus was found to be strongly modulated by extracellular potassium ([K+]out). When expressed in Xenopus oocytes, two jellyfish Shaker-like genes, jShak1 and jShak2, coding for potassium channels, exhibited similar modulation by [K+]out over a range of concentrations from 0 to 100 mM. jShak2-encoded channels also showed a decreased rate of inactivation and an increased rate of recovery from inactivation at high [K+]out. Using site-directed mutagenesis we show that inactivation of jShak2 can be ascribed to an unusual combination of a weak "implicit" N-type inactivation mechanism and a strong, fast, potassium-sensitive C-type mechanism. Interaction between the two forms of inactivation is responsible for the potassium dependence of cumulative inactivation. Inactivation of jShak1 was determined primarily by a strong "ball and chain" mechanism similar to fruit fly Shaker channels. Experiments using fast perfusion of outside-out patches with jShak2 channels were used to establish that the effects of [K+]out on the peak current amplitude and inactivation were due to processes occurring at either different sites located at the external channel mouth with different retention times for potassium ions, or at the same site(s) where retention time is determined by state-dependent conformations of the channel protein. The possible physiological implications of potassium sensitivity of high-threshold potassium A-like currents is discussed.
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INTRODUCTION |
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The large diversity of potassium channels
provides richness to the functional repertoire of excitable cells.
Their associated currents shape action potentials and regulate firing
frequency in addition to maintaining the membrane resting potential
(Connor and Stevens 1971; Hille 1992
).
The amplitude of the ionic current passing through the selective pore
of these channels is determined by channel conductance, the
transmembrane electrical field, and the potassium gradient. When the
extracellular potassium concentration ([K+]out) is increased,
the chemical driving force is reduced, which results in reduced
currents. In addition to this intrinsic property of all ionic channel
membranes, it has been shown that altering [K+]out produces a broad
spectrum of modulatory effects on the delayed rectifier current in
Xenopus axonal membrane (Safronov and Vogel 1996
), on the fast inactivating K+
current in rat hippocampal neurons (Pardo et al. 1992
),
and on heterologously expressed cloned representatives of the
Shaker subfamily of rapidly inactivating channels
(Baukrowitz and Yellen 1995
; Lopez-Barneo et al.
1993
; Tseng and Tseng-Crank 1992
). These modulatory effects include the following: regulation of the number of
channels available for activation, alterations in the rate of C-type
inactivation, and changes in frequency-dependent cumulative inactivation resulting from an interaction between N- and C-type inactivation. It has been suggested that these modulatory effects might
allow excitable cells to compensate for increases in
[K+]out in intercellular
space as a result of repetitive firing. Hounsgaard and Nicholson
(1983)
clearly demonstrated that an elevation of
[K+]out by as little as 1 mM may alter the pattern of spontaneous activity in guinea-pig Purkinje
neurons. Synchronous activation of a large population of these cells in
brain slices can raise the level of
[K+]out from 6 to 10 mM.
In higher vertebrates, extracellular potassium concentrations are regulated as a result of homeostatic mechanisms at the organ, tissue, and cellular levels. In contrast, there are no known organs or tissues maintaining relatively constant extracellular potassium levels in lower metazoans such as the cnidarians (hydroids, jellyfish, corals, etc). It is likely that neurons and other excitable cells in jellyfish have to be able to adapt to unstable extracellular potassium concentrations as a result of phasic accumulation of potassium during firing, or tonic changes due to naturally occurring salinity differences.
Despite their phylogenetic position at the base of the metazoans,
hydrozoan jellyfish display a variety of potassium channels and
currents (Meech and Mackie 1993; Przysiezniak and
Spencer 1994
). Several members of Shaker and
Shal subfamilies of genes encoding
-subunits of
voltage-gated potassium channels were cloned from the jellyfish
Polyorchis penicillatus. Their products, when expressed in
the Xenopus oocyte expression system, demonstrate biophysical properties similar to other known A-like currents (Jegla et al. 1995
; Jegla and Salkoff
1997
). The data presented in this paper demonstrate that
potassium channels in motor neurons from the jellyfish Polyorchis
penicillatus are modulated by
[K+]out as are two
heterologously expressed Shaker channels, jShak1 and jShak2, from Polyorchis. Modulation by
potassium is likely to be essential for nervous systems that experience
variable external concentrations of this ion. Increasing
[K+]out increased the
peak current amplitude for all three channels studied. We have shown
that occupation by K+ binding sites in the
external channel mouth in the closed state is crucial for modulation of
current amplitude by extracellular potassium. jShak2
channels exhibited strong potassium dependence of the inactivation rate
while recovery from inactivation was also affected by variations in
[K+]out.
Shaker potassium channels have at least two inactivation mechanisms: N- and C-type. N-type inactivation is associated with blockage of ionic current by the cytoplasmic N-terminus of the
-subunit of the channel protein, often called the "ball and
chain" mechanism of inactivation (Hoshi et al. 1990
;
Zagotta et al. 1990
). The molecular mechanism of C-type
inactivation is less clear, but it appears to be associated with
conformational changes in the external mouth of the channel
(Baukrowitz and Yellen 1995
; Liu et al.
1996
; Molina et al. 1997
).
Mutational analysis indicated that jShak2 channels show a strong, potassium-dependent C-type inactivation mechanism and that N-type inactivation is weak and "implicit." Enhancement of N-type inactivation of jShak2 by transplantation of a charged amino acid cluster from the N-terminus of the jShak1 channel sequence (jShak1 channels possess strong "explicit" N-type inactivation) rendered the inactivation rate less susceptible to variation of [K+]out. We suggest that an interplay between weak, "implicit," N-type inactivation and strong, and fast, C-type inactivation of jShak2 is responsible for the potassium-dependent cumulative inactivation observed in this channel.
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METHODS |
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Molecular biology
All jShak2 mutants were constructed using cassette,
PCR-based, site-directed mutagenesis as described previously
(Grigoriev et al. 1997). Mutants were verified by
sequencing in both directions using a Perkin-Elmer ABI 373A sequencer
and an ABI Prism Dye-Terminator Cycle Sequencing Kit. Construction of
the mutant jShak1
2-24 (jShak1T) was described
by Jegla et al. (1995)
. Capped mRNAs were prepared by
run-off transcription using mMessage mMachine kits (Ambion) for T3
(jShak1 and jShak1
2-24) or T7
(jShak2 and jShak2 mutants). The plasmid
containing a rat skeletal muscle sodium channel, the
-subunit gene,
rSkM1, was linearized with Sal I and transcribed using the
Ambion kit for T7.
Electrophysiological recording from swimming motor neurons
Primary cultures of swimming motor neurons of the jellyfish
Polyorchis penicillatus were prepared as described
previously (Przysiesniak and Spencer 1994) with the
modification that cells were exposed, with agitation, to collagenase
for only 1 h. Swimming motor neurons were identified by their
large size, clear cytoplasm, and a nucleus surrounded by membranous
structures. Whole cell patch recordings were made using 1-2 M
borosilicate glass pipettes filled with a solution that contained (in
mM) 500 KCl, 2 MgCl2,10 HEPES, 1 CaCl2, and 11 EGTA at pH 7.5 adjusted with
N-methyl glucamine (NMG). The extracellular bathing solution
contained (in mM) 450 NMG-Cl, 50 MgCl2, and 10 HEPES, at pH 7.5 adjusted with HCl. Potassium was introduced at the
indicated concentrations by equimolar NMG substitution. Cells were
microperfused using a manifold with a dead volume of <1 µl.
Solutions were completely exchanged within 2 s. All recordings
were carried out at room temperature (20-22°C).
Whole cell, two-electrode recording from Xenopus oocytes
Xenopus oocytes were prepared and injected with mRNA
as previously described (Grigoriev et al. 1997). mRNA
(1-5 ng) was injected in each oocyte using a volume of 50 nl. The
amount of injected RNA was adjusted for each expressed channel type to
minimize the effects introduced by high levels of channel expression
(Grigoriev et al. 1999
). Whole cell currents were
recorded between 2 and 3 days after injection using a
two-microelectrode voltage clamp (CA-1, Dagan, Minneapolis, MN). Cells
were constantly microperfused with a gravity fed system.
Outside-out macropatch recording from Xenopus oocytes
Outside-out macropatches were obtained and recordings made as
described by Stühmer et al. (1992). Patch
recordings, as well as whole cell patch recordings from swimming motor
neurons, were made using an Axopatch 1D amplifier (Axon Instruments).
Fast perfusion was via two PE tubes (7405, Intramedic) glued to the
bottom of a 35 mm plastic Petri dish with their orifices perpendicular
and in contact with one another. The two resulting perfusion streams were deflected where they met and then ran parallel to one another. Because of the small dimensions and relatively low flow rates, the
perfusion system was operating at a low enough Reynold's number as to
produce laminar flow. Because NMG substituted for potassium, it was
possible to visualize an optical density difference between the K
mM = 0 mM and the K mM = 100 mM streams under phase contrast optics, and mixing was not seen at the boundary between the two solutions. Each of these tubes was connected to a reservoir filled with
the relevant solution. These reservoirs were separately connected to a
pair of Picospritzers (General Valve Corporation, Fairfield, NJ) that
were controlled by a computer. At the beginning of each recording
session, control and test solutions were perfused at 0.5 cm
s
1 by gravity. Pipettes containing the
macropatch were positioned closer to the orifice of the tube containing
the control solution than the test solution. Pressure pulses (1 bar)
applied to the reservoir containing the test solution by a Picospritzer
increased the velocity of the test solution to 5 cm
s
1, thereby deflecting the control stream and
exposing the macropatch to the test solution. Solutions could be
changed in this way within 1 ms. The extracellular solution without
potassium, [K+]out = 0, contained 100 mM NMG-Cl, 3 mM MgCl2, 10 mM
HEPES-acid adjusted to pH 7.5; whereas the solution,
[K+]out = 100 mM
contained KCl 95 mM, MgCl2 3 mM, 10 mM
HEPES/1/2K adjusted to pH
7.5. Intermediate concentrations of K+ were made
by mixing these two solutions in the required proportion. The
intracellular solution contained (in mM) 100 KCl 100, 3 MgCl2, 10 EGTA, and 10 HEPES adjusted to pH 7.5. Experiments were carried out at 20°C using a temperature controller
TC-10 (Dagan, Minneapolis, MN). Conductance calculations for whole
oocyte recordings were made using intracellular potassium activity of
147 mM as reported by Kusano et al. (1982)
.
Data acquisition and experimental control
All data acquisition and experimental control was achieved with a Digidata 1200 acquisition system (Axon Instruments, Foster City, CA) running pClamp 6.1 software (Axon Instruments). Analysis and fitting of experimental data were done using the Clampfit program of the pClamp 6.1 suite and SigmaPlot 4.00 (SPSS, Chicago, IL). All results are expressed as means ± SE.
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RESULTS |
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External potassium modulates A-like currents in identified neurons and heterologously expressed jellyfish Shaker channels
Recordings from cultured, identified, motor neurons that control
swimming demonstrate the presence of two potassium currents; a fast
inactivating A-like current, IK-fast,
and a delayed rectifier, IK-slow
(Przysiezniak and Spencer 1994). In this study we were able to show that IK-fast current was
strongly modulated by
[K+]out (Fig.
1). Eliminating potassium ions from the
external solution completely inhibited this current (Fig. 1,
inset), leaving a "potassium insensitive," slowly
activating current, IK-slow.
Increasing [K+]out from 1 to 100 mM amplified the peak, whole cell conductance of channels
passing IK-fast more than threefold
(Fig. 1). We were able to demonstrate the potassium insensitivity of
IK-slow by using a holding potential
of
40 mV, which completely inactivated IK-fast (Przysiezniak and
Spencer 1994
), and then altering
[K+]out from 1 to 100 mM
(data not shown).
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Jellyfish jShak1 and jShak2 genes encode
high-threshold, A-like currents (Jegla et al. 1995).
When expressed in Xenopus oocytes, jShak1 and
jShak2 currents showed a strong dependence on the
concentration of extracellular potassium ions (Fig.
2, A and B). When
[K+]out was increased
from 1 to 100 mM, the conductance for both channels increased by
approximately fourfold and the current by 33%; we call this the
amplitude effect. The effect of increasing [K+]out on the
conductances of jShak1 and jShak2 could be fitted by a combination of an equation similar to a Michaelis-Menten relationship, having an apparent Km of
~1.5 mM, and a linear relationship with a slope of 0.005 mM
1 (Fig. 2C). We suggest that this
nonhyperbolic component represents a low-affinity mechanism that
appears as a linear slope within the range of concentrations used.
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With increasing concentrations of external K+,
jShak2 inactivated more slowly (Fig. 2D, = 15.67 ± 0.27 ms and 71.81 ± 7.75 ms at
[K+]out = 1 mM and 100 mM, respectively, n = 9). This change in
with
alterations in [K+]out
was not seen in either jShak1 (Fig. 2D) or
IK-fast (Fig. 1, inset).
The time constant of IK-fast
inactivation was not significantly different (P = 0.45, n = 5) between 1 and 100 mM
[K+]out.
Several monovalent cations, other than K+,
produced qualitatively similar effects on jShak2 current,
but with substantially lower efficacy than potassium ions. Their
modulatory effectiveness for both current amplitude and inactivation
kinetics could be ranked as follows: K+ > Rb+ > Cs+ > Na+ (data not shown). A similar sequence of
sensitivity of current peak amplitude to extracellular monovalent
cations was demonstrated for the rat neuronal channel, RCK4 or Kv1.4
(Pardo et al. 1992).
Modulatory effects of [K+]out involve N-type inactivation
It is well established that N-type inactivation in
Shaker potassium channels involves a "ball and chain " mechanism (Hoshi et al. 1990; Zagotta et al.
1990
). Deletion of the cytoplasmic N-terminus of the
Shaker channel protein eliminates fast (N-type) inactivation
revealing a slow (C-type) inactivation that has been associated with
conformational changes in the external mouth of the channel
(Baukrowitz and Yellen 1995
; Liu et al.
1996
; Molina et al. 1997
).
Extracellular potassium strongly affects the inactivation rate of
jShak2 but not of jShak1 currents, which may
reflect differences in the molecular mechanism of inactivation. It is
known that partial blockade of Shaker channels by
extracellular application of TEA is accompanied by a concomitant
slowing down of C-type inactivation. Conversely, rapid N-type
inactivation is TEA-insensitive (Choi et al. 1991;
Grissmer and Cahalan 1989
). Extracellular administration of TEA slowed down the rate of inactivation of jShak2 but
not fast inactivation of jShak1 (Fig.
3), indicating that the mechanisms of
inactivation of jShak1 and jShak2 differ.
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To examine N- and C-type inactivation mechanisms more closely, we
constructed mutants with modified N-termini (Fig.
4). A previous study (Jegla et al.
1995) showed that deletion of the first 23 residues of
jShak1 yielded channels lacking fast inactivation (Fig.
5A). This mutant,
jShak1
2-24, showed noticeable conductance even in the
absence of external potassium. In the absence of
[K+]out there were two
distinct components of activation of jShak1
2-24, slow
and fast. Presumably the slow component resulted from additional activation of the channels by potassium effluxing through the already
open channels and accumulating in the intermicrovillar space of the
oocyte (Grigoriev et al. 1999
). Dose-response curves suggest an
increase in the apparent affinity of this mutant channel to external
potassium: the K value for gapp of
jShak1
2-24 was 0.2 mM compared with 1.5 mM for Wild-type
jShak1 (Fig. 5, A and C). A truncated
jShak2 mutant, (jShak2
2-38) was not as
sensitive to [K+]out,
having a Kgapp of ~2 mM (Fig. 5, B
and C). The effect of N-terminal deletion on the
inactivation rate of jShak2 was not pronounced (Fig. 5,
B and D), with statistically significant
differences only being observed for concentrations of
[K+]out > 30 mM.
Although the results obtained for jShak1
2-24 are consistent with a "ball and chain" mechanism, the data obtained for
jShak2
2-38 are more difficult to associate with such a
mechanism and suggest that inactivation is due to rapid, C-type
inactivation. To determine whether any N-type inactivation is present
in jShak2, we compared the rates of recovery from
inactivation in both jShak1 and jShak2 and their
N-terminal deleted mutants using a two pulse protocol (Fig.
6). Recovery from inactivation of Wild
jShak2 could be fitted with the sum of slow (
= 1 s) and fast (
= 0.1 s) exponents, suggesting that
there are two distinct processes involved in inactivation. The relative
contribution of the slow and fast components depended on
[K+]out, such that
increasing the external potassium concentration increased the
proportion of the fast process. Although the jShak2
2-38 mutant also demonstrated potassium dependence, recovery from
inactivation became monoexponential, indicating the presence of only
one mechanism. Disappearance of the slow-recovering component of the
jShak2 current after N-terminal truncation shows that this
part of the protein is involved in the inactivation process. Thus the
presence of the second component of recovery in Wild-type
jShak2 and its absence in jShak2
2-38 together
with the absence of a pronounced change in the inactivation rate at low
[K+]out leads us to the
assumption that jShak2 experiences both weak, N-type
inactivation and strong, fast, C-type inactivation. It is important to
note that, under similar ionic conditions, C-type inactivation in
Drosophila Shaker channels was two orders of magnitude slower than in jShak2 (Baukrowitz and Yellen
1995
).
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Comparisons of the sequences of jShak1, jShak2,
and fly Shaker N-termini indicate some structural
conservation (Jegla et al. 1995); however, there are
differences in the number and distribution of charged amino acids in
the first 25 amino acids (Fig. 4). jShak1 carries two
distinct clusters of charge on its N-terminus: RRKKE with a strong net
positive charge, and KDDE with a net negative charge. Such pronounced
clusters are not present in the N-terminal region of jShak2.
It was shown that reducing the charge on the N-terminus of fly
Shaker channels by incremental shortening of the N-terminus
alters the rate of inactivation (Zagotta et al. 1990
).
Furthermore, structural analysis of the N-termini of RCK4 and Raw3
showed that negatively and positively charged residues may be
distributed in such a way as to confer dipole properties on the ball
(Antz et al. 1997
). Therefore it is possible
that the transmembrane electrical field orients the ball in the
cytoplasmic mouth of the channel during the inactivation process. It is
also possible that the presence of the charged residues in the
N-terminus sequence might be required for its effective interaction
with the ball receptor located in the internal channel mouth. The
presence of electrostatic and steric interactions has been suggested
for the ball peptide and the ball receptor (Holmgren et al.
1996
).
To examine the influence of these clusters of charged residues on the
sensitivity of inactivation kinetics to extracellular potassium, we
constructed mutants of jShak2 (Fig. 4) in which positively (RRKKE) and negatively charged (KDDE) clusters were transplanted from jShak1 to the jShak2
N-terminal region, both separately (jShak2N+
and jShak2N) and together
(jShak2N+/
). Introduction of a positively
charged cluster reduced the influence of
[K+]out on the rate of inactivation, whereas
inclusion of a negatively charged cluster of amino acids enhanced the
potassium dependency of this phenomenon (Fig.
7A). The decrease in
potassium sensitivity of inactivation shown by
jShak2N+ was accompanied by a far slower
(almost 100-fold) rate of recovery from inactivation (Fig.
7B). Conversely, addition of a negative cluster slightly
increased the rate of recovery (Fig. 7B). Inclusion of
both charge clusters (jShak2 N+/
) had
intermediate effects on both the potassium sensitivity of the
inactivation rate and the time for recovery from inactivation (Fig.
7B).
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The effectiveness of the N-terminal region as an inactivation
particle can be judged from the rate of recovery from inactivation. Charge-dependent changes in the recovery rates seen in N-terminal mutants probably reflect differences in the strength of interaction between the inactivation particle and presumed receptor sites in the
internal channel mouth. Figure 7C shows the effect of
these mutations on the effectiveness of
[K+]out modulation of both peak current and
the inactivation time constant. Altering the effectiveness of the
"ball and chain" mechanism did not significantly change the
amplitude effect of [K+]out, but these
mutations did noticeably alter the sensitivity of the rate of
inactivation to the extracellular potassium concentration. For example,
when N-type inactivation was more effective
(jShak2N+), the external potassium
concentration no longer had a strong influence on the inactivation
rate. Decreasing the effectiveness of N-terminal inactivation
(jShak2N) had the opposite effect.
Compared with the Wild-type, there was an additional increase in the
sensitivity of the inactivation mechanism to
[K+]out. The mutant
jShak2N+/
demonstrated an intermediate
sensitivity of the inactivation rate to
[K+]out.
Fast perfusion experiments reveal differences between amplitude and inactivation effects of [K+]out
When patch recordings of jShak2 current were made in
the outside-out configuration at a high external
[K+] (100 mM), there was a noticeable increase
in the inactivation rate ( = 12.8 ± 1.3 ms,
n = 6) compared with whole cell recordings (
= 71.8 ± 7.7 ms, n = 6; Fig.
8A). We suggest that this
phenomenon is associated with the accumulation of potassium ions in
intermicrovillar space close to the oocyte membrane during channel
activation when making whole cell recordings. The original microvillar
structure is not preserved in outside-out patch recordings, thus
eliminating many of the barriers to diffusion of potassium ions as they
efflux through the pore (Grigoriev et al. 1999
).
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Using a fast perfusion system (solution exchange in <1 ms), we
were able to detect differences in the number of channels available on
depolarization and in their rates of inactivation when
[K+]out was rapidly
altered (Fig. 8A). Channels that had been activated instantly changed their time constants of inactivation from 12.8 ± 1.3 ms (n = 6) to 3.5 ± 0.3 ms
(n = 6) following a rapid switch from 100 to 0 mM
[K+]out. In Fig.
8A the amplitude effect is seen as a decrease in the peak
amplitude of currents recorded at increasing intervals after switching
from 100 to 0 mM. The rate of reduction in the peak current reflects
the rate of elimination of potassium from the channel mouth in the
resting state. This removal of [K+] from the
channel mouth by diffusion (dekalification) occurred more slowly in the
resting state ( = 13 ms) than in the open state when the rate
of dekalification is comparable with the speed of switching between
solution (i.e., <1 ms). These results indicate that both the amplitude
and inactivation effects of
[K+]out are due to
processes occurring at either different sites with different retention
times for potassium ions or at the same site where retention time is
determined by conformation of the channel protein. Experiments in which
dekalification of channels occurred after opening indicate that, as a
result of losing potassium, they were converted to a state from which
recovery was slow. This happened in spite of a high
[K+]out being restored
rapidly after inactivation (Fig. 8B).
Physiological implications of channel sensitivity to [K+]out
Although the data presented above showed that
[K+]out can regulate both
the conductance and inactivation rate of jShak2, we wanted
to determine whether such modulation of channel properties could modify
the excitability properties so as to be physiologically significant. We
were able to produce synthetic action potentials by co-expressing
jShak2 and the rat skeletal muscle sodium channel -subunit, rSkM1 (Fig.
9A). These action potentials
repolarized with two distinct phases: a rapid, early phase provided by
the A-like properties of jShak2, followed by a slow phase
presumably associated with an inward rectifier current endogenous to
Xenopus oocytes (Bauer at al. 1996
).
Increasing [K+]out from 1 to 40 mM in the presence of constant
[Na+]out caused an
increase in the rate of early repolarization from 73.3 ± 2 s
1 (n = 4) at
[K+]out = 1 mM to
86.7 ± 4.5 s
1 (n = 4) at
[K+]out = 40 mM
(P = 0.034) and an associated exaggeration of the plateau phase. Increasing
[K+]out caused late
repolarization to become much slower and the spike broader as a result
of the decreased electrochemical driving force on
K+ through endogenous inward rectifier channels.
In these experiments the decreased resting potential resulting from an
increase in [K+]out was
compensated by adjusting the holding current in current clamp mode.
Inhibition of jShak2 current by application of 2 mM 4-aminopyridine (4-AP) decreased the rate of early repolarization and
positioned the plateau closer to the level of the sodium reversal potential. It should be noted that the endogenous inward rectifier current in oocytes is not sensitive to 4-AP (Bauer et al.
1996
). We suggest that this modulation by
[K+]out could stabilize
the plateau phase of action potentials when potassium accumulates in
restricted extracellular spaces during repetitive firing.
|
Another mechanism that could counteract the effect of potassium
accumulation on the driving force and hence action potential shape is
the inhibitory effect of
[K+]out on the cumulative
inactivation of jShak2 in the course of repetitive
stimulation (Fig. 9B). Elevation of extracellular potassium concentration makes cumulative inactivation of channels less
pronounced. The severe reduction of cumulative inactivation observed
for the jShak2 2-38 mutant suggests that the N-terminal
"ball" might play a pivotal role in this process.
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DISCUSSION |
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Figure 10 is a kinetic model for both jShak1 and jShak2 and provides a background for the discussion.
|
Jellyfish A-like current (IK-Fast)
recorded from swimming motor neurons and currents in oocytes
expressing jShak1 and jShak2 showed modulation of
the peak current amplitude by altering the external potassium
concentration. Only jShak2 experienced modulation of its
inactivation rate by changes in
[K+]out. An effect of
external [K+ ] on current amplitude has been
reported for other potassium channels, such as the delayed rectifier in
Xenopus axonal membrane (Safronov and Vogel
1996), heterologously expressed mammalian neuronal RCK4
channels (Pardo et al. 1992
), and the T449K mutant of
fly Shaker (Lopez-Barneo et al. 1993
). RCK4
channels in the absence of external potassium became nonconducting,
although gating currents could still be recorded, indicating that the
voltage-sensing mechanism remains operational. Mutation of the pore
region of the fly Shaker channel protein by substitution of
tyrosine residue 449 with lysine made peak current amplitude of this
channel strongly dependent on
[K+]out
(Lopez-Barneo et al. 1993
). It was suggested that
potassium ions, as well as other monovalent cations, can occupy site(s) in the external channel mouth preventing development of C-type inactivation by a "foot in the door" mechanism (Labarca and
MacKinnon 1992
; Lopez-Barneo et al.
1993
). If depolarization occurs in the absence of
[K+]out, channels proceed rapidly to the
C-type inactivated state after opening (Fig. 10). C-type inactivation
occurs sufficiently rapidly so as to convert many K+
channels to a nonconducting state before any significant current can be
recorded and before sites in the mouth become occupied by potassium
effluxing from the cytoplasm. Current can only be detected when C-type
inactivation is slowed sufficiently by extracellular potassium ions. An
efficient ball and chain mechanism, as was seen for
jShak1 in this study, prevents occupation of the
proposed potassium binding site(s) by effluxing potassium ions, which
can explain the increased apparent affinity to
[K+]out observed for the N-terminus truncated
mutant. The "foot in the door" hypothesis for modulation by
[K+]out also provides an explanation for the
effect of [K+]out, and other monovalent
cations, on inactivation of jShak2. Inactivation of this
channel occurred predominantly by a C-type mechanism, with occupation
of the potassium binding site(s) slowing inactivation. The differences
in retention times of K+ for closed and open channels
observed in experiments involving fast dekalification probably reflects
state-dependent conformational changes, or accessibility, of potassium
binding site(s) located in the external channel vestibule. N-type
inactivation of jShak2 channels is "implicit," but
experiments examining recovery from inactivation of Wild-type and
jShak2
2-38 unmasks the presence of an N-type
inactivation mechanism. Its presence can also be detected at
[K+]out of >30 mM when C-type inactivation
becomes very slow (Fig. 5D). Conversely, inactivation of
jShak1 is mostly by an efficient ball and chain
mechanism (Fig. 10). In the case of jShak2, enhancement of N-type inactivation by transplantation of the jShak1
RRKKE cluster (jShak2N+) makes the potassium
dependence of inactivation less pronounced because the
potassium-independent ball and chain mechanism becomes more explicit.
According to the kinetic model suggested by Baukrowitz and
Yellen (1995) for fly Shaker channels, recovery
from N-type inactivation is fast, whereas recovery from C-type
inactivation is much slower. Interaction between slowly recovering,
potassium-dependent, C-type inactivation and rapidly recovering,
potassium-independent N-type inactivation explains the potassium
dependency of cumulative inactivation observed for fly
Shaker channels. In our experiments with
jShak2 channels, the slower component of recovery
appears to be tightly associated with N-type inactivation. Truncation
of jShak2 channels was accompanied by disappearance of
the slower component of recovery, and introduction of the RRKKE cluster
in the N-terminal sequence made recovery dramatically slower. It is
reasonable to suggest that the increase in effectiveness of interaction
between the inactivation particle and the receptor in the internal
channel mouth simultaneously slows down the process of unbinding of
this particle from the receptor during recovery from inactivation. Our
data indicate that N-type inactivation in jShak2
channels is the major mechanism involved in potassium-dependent
cumulative inactivation.
At the end of a depolarizing pulse, jShak2 channels can
be in N, C, and C + N inactivated states (Fig. 10).
jShak2 channels with the N-terminus present show
potassium-sensitive recovery from inactivation indicating that channels
in the C + N inactivated state recover more slowly than those in the N
state. Why do channels in the C + N state recover slowly and N-type
inactivated channels recover rapidly? Displacement of the N-particle
from the internal channel mouth will allow the channel to proceed to
the resting state and determine the rate of recovery from inactivation.
Considering that impermeable Cs+ ions (data not shown) can
imitate the effect of K+ on the process of recovery from
inactivation in jShak2 then occupancy of the site(s) at
the external channel vestibule, rather than ion flow through the
reopened channels, can explain this effect. Similarly,
Gomez-Lagunas and Armstrong (1994) reported that
Cs+ could substitute for K+ in the process of
recovery of Shaker B channel from inactivation. There
are at least two possible mechanisms by which occupation of this
site(s) can promote displacement of the inactivation ball. One
explanation involves electrostatic interaction between ion(s) occupying
site(s) in the external channel mouth and the inactivation ball
(Gomez-Lagunas and Armstrong 1994
). Another suggested
mechanism is that occupation of the K+ binding site
prevents the conformational changes associated with C-type
inactivation. The latter mechanism assumes that such conformational changes can have a remote influence on more internalized parts of the
protein participating in ball-receptor interaction. The ultimate effect
of the conformational change is to hold the inactivation particle in
place for a longer time. This mechanism can also explain the absence of
visible jShak2 tail currents at low concentrations of
[K+]out, as seen in Fig. 2B,
because most of the inactivated channels are in a C + N type
inactivated state and channel reopening after repolarization
(Ruppersberg et al. 1991
) cannot be observed.
Both jShak2 and fly Shaker exhibit
potassium-dependent cumulative inactivation. This dependence was
explained by Baukrowitz and Yellen (1995) by assuming
that there is an interplay between an "explicit" N-type mechanism
and slow C-type inactivation. However, for jShak2
channels, we suggest that the potassium dependence of cumulative
inactivation is caused by a combination of "implicit" N-type
inactivation and fast C-type inactivation.
All Polyorchis potassium-dependent currents, such as
jShak1, jShak2 and
IK-fast, show a high activation threshold
that is associated with their roles in shaping the early repolarization of the action potential. We also observed that when
jShak2 contributes outward current in synthetic action
potentials expressed in oocytes, this current is capable of both
repolarizing the action potential and forming a plateau. The endogenous
repolarizing outward current in swimming motor neurons of
Polyorchis, IK-fast, also
truncates the plateau of action potentials, which has been shown to
modulate neuromuscular transmission (Przysiezniak and Spencer
1994; Spencer 1984
; Spencer et al.
1989
). Jellyfish have no known tissues or cells, such as glia,
that are specialized for K+ homeostasis in the immediate
extracellular space surrounding neurons. It is also important to note
that nearly all cell types in hydromedusae are electrically excitable,
including epithelial cells (Satterlie and Spencer 1987
),
which would drastically increase the number of potential sources for
potassium accumulation in extracellular space. Therefore it is likely
that potassium that accumulates during repetitive firing could reduce
outward current amplitude and duration, thereby altering action
potential shape. Because IK-fast can be
modulated by [K+]out, one can imagine that
these negative influences by accumulating K+ can be
compensated by increased current amplitude. Although the kinetics and
electrical properties of IK-fast are
markedly different from those of heterologously expressed
jShak1 and jShak2 currents, we cannot
rule out the possibility that IK-fast is
conducted by channels composed of jShak1 or
jShak2
-subunits because their properties may be
altered by differences in lipid environments (Schetz and
Anderson 1993
) and/or by the presence of auxiliary subunits
when these channels are expressed in vivo.
In the mammalian CNS, extracellular potassium concentrations can
increase by several millimoles as a result of high-frequency firing
(Hounsgaard and Nicholson 1983; Sykova
1983
). It is probably significant that the potassium
sensitivity shown by various vertebrate (Pardo et al.
1992
; Safronov and Vogel 1996
) and fly
(Baukrowitz and Yellen 1995
) K+ channels can
be satisfactorily described by curves with a Kapp close to
2 mM. By comparison, there are additional low-affinity sites that are
modulating potassium currents in Polyorchis. These sites
are presumably an adaptation for the lack of glia cells and the
consequently greater potassium accumulation in extracellular space.
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ACKNOWLEDGMENTS |
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We thank Bamfield Marine Station for providing excellent facilities. We are especially grateful to W. Gallin for advice on molecular techniques and P. Ruben for providing the plasmid containing the rSkM1 channel gene. We are also grateful to T. Baukrowitz, P. Ruben, and S. Buckingham for stimulating discussion and valuable advice. We especially thank Dr. W. Gallin for providing technical guidance for the molecular biological aspects of this study, which were carried out in his laboratory.
Partial salary support for N. G. Grigoriev was provided by the Western Canadian Universities Marine Biological Society. J. D. Spafford was supported by an Alberta Heritage Foundation for Medical Research studentship. This work was supported by a Natural Sciences and Engineering Research Council Research grant to A. N. Spencer.
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FOOTNOTES |
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Address for reprint requests: A. N. Spencer, Bamfield Marine Station, Bamfield, British Columbia V0R 1B0, Canada.
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. Section 1734 solely to indicate this fact.
Received 30 March 1999; accepted in final form 16 June 1999.
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REFERENCES |
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