(Received for publication, March 7, 1997, and in revised form, April 1, 1997)
From the Department of Physiology and Pharmacology, Sackler School
of Medicine, Tel-Aviv University, 69978 Ramat Aviv, Israel and the
Department of Physiology and Biophysics, Mount Sinai
School of Medicine, The Mount Sinai Hospital, New York, New
York 10029-6574
Kv1.1/Kv1.1 (
)
K+ channel expressed in Xenopus oocytes
was shown to have a fast inactivating current component. The fraction of this component (extent of inactivation) is increased by
microfilament disruption induced by cytochalasins or by phosphorylation
of the
subunit at Ser-446, which impairs the interaction of the
channel with microfilaments. The relevant sites of interaction on the channel molecules have not been identified. Using a
phosphorylation-deficient mutant of
, S446A, to ensure maximal basal
interaction of the channel with the cytoskeleton, we show that one
relevant site is the end of the C terminus of
. Truncation of the
last six amino acids resulted in
channels with an extent of
inactivation up to 2.5-fold larger and its further enhancement by
cytochalasins being reduced 2-fold. The wild-type channels exhibited
strong inactivation, which could not be markedly increased either by cytochalasins or by the C-terminal mutations, indicating that the
interaction of the wild-type channels with microfilaments was minimal
to begin with, presumably because of extensive basal phosphorylation.
Since the C-terminal end of Kv1.1 was shown to participate in channel
clustering via an interaction with members of the PSD-95 family of
proteins, we propose that a similar interaction with an endogenous
protein takes place, contributing to channel connection to the oocyte
cytoskeleton. This is the first report to assign a modulatory role to
such an interaction: together with the state of phosphorylation of the
channel, it regulates the extent of inactivation conferred by the
subunit.
In contrast with the well understood structural determinants within the core of voltage-gated K+ (Kv) channel proteins that underlie many functional properties (e.g. voltage-sensing regions, pore region; Refs. 1-5), much less is known about the role of extrinsic factors in determining Kv channel function. One such factor is the interaction of ion channels with the cytoskeleton, which could affect structural and functional organization of neuronal elements. Recently, a family of membrane-associated putative guanylate kinases were shown to bind directly to K+ channels of the Shaker (Kv1) family and to induce their clustering in COS7 cells (6, 7). This family includes PSD-95, an abundant synaptic protein found both pre- and postsynaptically, and SAP97, a protein found in axons of neurons as well as in non-neuronal cells (for reviews, see Refs. 8 and 9). These data together with the demonstration of colocalization of the channels with PSD-95 (6, 10) in the brain, particularly in nerve terminal plexuses of basket cells in the cerebellum, provide correlative evidence of a direct association in vivo. Apart from a probable role in clustering, it is not known whether the association with PSD-95 has a functional role of modulating the biophysical characteristics of the channel.
For several years now our interest has been the modulation by direct
phosphorylation of the voltage-gated K+ channel Kv1.1
(originally cloned from rodent brain cDNA libraries; Refs. 11-14),
which is expressed in Xenopus oocytes as a delayed rectifier
type (15, 16). Recently it became clear that part of the diversity of
Kv channels may arise from association of pore-forming Kv1
subunits with auxiliary Kv subunits (for review, see
Ref. 17) and that the functional consequence of the association between
Kv1.1 (
) and rat brain Kv
1.1 (
) is the
appearance of rapid inactivation of the current (18-20). Therefore, we
studied also the Kv1.1/Kv
1.1 (
) channel and its modulation by
direct phosphorylation (21). We demonstrated that the inactivation of
the
current is not complete, even under conditions where the
polypeptide is saturated with the
polypeptide, and has an inherent
sustained component, indistinguishable from a pure
current. The
extent of the inactivation (the fraction of the inactivating component)
is increased either by phosphorylation of the
subunits at Ser-446
or by depolymerization of the microfilaments by cytochalasins; the
latter effect occludes the former. To account for our findings we
proposed a simple model that assumes the existence of two modes of the
channels, in one mode inactivation is conferred on the channels
by the
, in the other mode no inactivation is observed despite the
fact that
is physically associated with
. Interaction of the
channels with microfilaments shifts the equilibrium between the two
modes toward the noninactivating mode, and phosphorylation (which
impairs the interaction of the channels with microfilaments by an
unknown mechanism) shifts the equilibrium toward the inactivating mode.
The
protein has at its very C terminus a TDV sequence (amino acids
493-495), which presumably mediates the coclustering of Kv1.1 channels
with PSD-95 and SAP97 in COS7 cells (7). Thus, we assumed that this
site might mediate the interaction of the
channels with the
oocyte microfilaments; phosphorylation of Ser-446, which is also in the
cytoplasmic C terminus of the
protein, seemed an attractive
mechanism to modulate this interaction. Supportive of such a notion is
the recent demonstration that the binding of the inwardly rectifying
K+ channel Kir 2.3 to PSD-95 is regulated by
phosphorylation of the channel; however, in this case the
phosphorylation occurs at the binding site itself (22).
In this study we set out to test the site of interaction with the
cytoskeleton by studying the effect of truncation of the last
C-terminal amino acids of the protein on the extent of inactivation
of the phosphorylated and nonphosphorylated
channels and their
susceptibility to microfilament depolymerization. The results are in
agreement with a scenario where the very C terminus of the
subunits
in
channels interacts with the microfilaments via a PSD-like
protein; the interaction is phosphorylation-dependent and
affects the extent of channel inactivation.
Chemicals were from Sigma (Rishon Le-Zion,
Israel) unless stated otherwise. Vanadate (sodium orthovanadate) and
okadaic acid were from Alomone Laboratories (Jerusalem).
[35S]Methionine/cysteine mix and
[-32P]ATP were from Amersham Corp. Kv1.1
antiserum was generated against a 23-amino acid peptide that
corresponds to the N terminus of RCK1 (SGENADEASAAPGHPQDGSYPRQ), as
described (15).
Kv1.1 cDNA was
subcloned into a vector to yield a SupEx-RCK1 construct that confers
high levels of expression in oocytes (15). All substitution mutants of
Kv1.1 were subcloned into the same vector.
Oligonucleotide-mediated mutagenesis was performed using a mutagenesis
kit (CLONTECH) according to the manufacturer's
instructions, using double-stranded DNA as templates and
oligonucleotide primers (synthesized by General Biotechnology Inc.,
Rehovot, Israel) encoding the desired mutations. S446A was generated as
described (16); K490s.c and T493A were generated on either the
WT1 channel or S446A mutated molecules
using the primers (underlined nucleotides encode the mutated residues):
5
-CGTTAATAAGAGCTAGCTCCTGACCG-3
; 5
-GCAAGCTCCTGGCCGATGTTTAAAAAAAGC-3
, respectively.
mRNAs were transcribes in vitro using T7 RNA
polymerase.
Frogs (Xenopus laevis) were maintained and
dissected, and their oocytes were prepared as described (23). Oocytes
were injected with 2-5 ng/µl Kv1.1 and 200-500 ng/µl
Kv1.1 mRNA for two electrode voltage clamp studies
and 100-200 ng/µl Kv1.1 and 3-7 µg/µl
Kv
1.1 mRNAs for macropatch and biochemical studies.
The concentrations of the injected mutant RCK1 mRNAs deviated
slightly from the above concentrations as they were adjusted to give
similar current amplitudes with Kv
1.1. Injected oocytes
were incubated at 22 °C for 1-3 days in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 5 mM
Hepes, pH 7.5) supplemented with 2.5 mM sodium pyruvate, 100 µg/ml streptomycin, and 100 units/ml penicillin (NDE solution) and then assayed either electrophysiologically or biochemically. Cytochalasins were added 4-6 h before the electrophysiological assay.
Two electrode voltage clamp recordings were performed as described
(16). To avoid possible errors introduced by series resistance, only
current amplitudes up to 4 µA were recorded, and in a given experiment the amplitudes of WT and mutant currents were similar. Oocytes were placed in a 1-ml bath continuously perfused with ND96
solution. Currents were elicited by stepping the membrane potential
from a holding potential of 80 mV to +50 mV for 70 ms. Net current
was estimated by subtraction of the scaled leak current elicited by a
voltage step to
90 mV.
This was done essentially as described (16). Following injection, six to eight oocytes were incubated at 22 °C for 4 h in NDE solution, then for 3 days in NDE containing 0.2 mCi/ml [35S]Met/Cys, and then were homogenized in 150-300 µl of medium consisting of 20 mM Tris, pH = 7.4, 5 mM EDTA, 5 mM EGTA, 100 mM NaCl, 50 µg/ml phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, 1 µM pepstatin, 1 mM 1,10-phenanthroline supplemented with protein phosphatase inhibitors: 50 nM okadaic acid, 0.5 mM vanadate, and 50 mM KF. Debris was removed by centrifugation at 1000 × g for 10 min at 4 °C. After addition of Triton X-100 to a final concentration of 4%, followed by centrifugation for 15 min at 4 °C, antiserum was added to the supernatant for 16 h at 4 °C. The antibody-antigen complex was incubated for 1 h at 4 °C with protein A-Sepharose and then pelleted by centrifugation for 1 min at 8000 × g. Immunoprecipitates were washed four times with immunowash buffer (150 mM NaCl, 6 mM EDTA, 50 mM Tris, pH = 7.5, 0.1% Triton X-100); the final wash contained no Triton X-100.
Quantification of Labeling Intensities and Generation of Digitized PhosphorImager ScansGels were dried and placed in a PhosphorImager (Molecular Dynamics) cassette for about 1 day. Using the software ImageQuant, a digitized scan was derived, and relative intensities of protein bands were estimated quantitatively by the software ImageQuant as described (16).
Statistical AnalysisData are presented as means ± S.E.; N denotes number of frogs assayed, n denotes the number of oocytes assayed. The Student's t test was used to calculate the statistical significance of differences between two populations.
The mRNAs of Kv1.1 () and Kv
1.1 (
) were coinjected at
a
-to-
mRNA ratio of 100, which ensures saturation of
polypeptides with
. As described before (21), the resulting outward
current assayed by the two-electrode voltage clamp technique had a fast inactivating component (Ii) and a noninactivating sustained
component (Is) remaining at the end of a depolarizing pulse
to +50 mV. The apparent extent of inactivation (inactivating fraction)
was defined as Ii/Ip (Ip = peak
current). Previously, we showed that the extent of inactivation of the
S446A
channel (in which Ser-446 of the
subunit
was substituted with alanine; S446A) is significantly smaller (by about
70%) than that of the WT (
WT
) channel and related it
to the fact that the mutant, unlike the wild-type, channel is not
phosphorylated in its basal state in the oocyte (21). In this study the
average extent of inactivation of
WT
currents was
0.57 ± 0.02 (mean ± S.E.; N = 3 oocyte
batches, n = 41 oocytes) and that of
S446A
currents was 0.12 ± 0.025 (N = 6 oocyte batches, n = 78 oocytes).
Truncation of the very end of the C terminus of S446A polypeptide
by deletion of the last six amino acids (substituting Lys-490 with stop
codon: K490s.c), or substitution of Thr-493 with Ala (T493A), increased
by 155 and 65% the extent of inactivation of the respective
S446A
channels (see Fig. 1,
A and B, open bars). The same mutations in the WT
polypeptide did not increase the extent of inactivation of the
corresponding
channels (Fig. 2, A and
B, open bars). Previously, we showed that treatment of oocytes with 40 µM of the microfilament-disrupting agent
dihydrocytochalasin B (DHCB) increases the extent of inactivation, the
effect being severalfold larger in the
S446A
than in
the
WT
channels. If, as we believed, the above
increases in the extent of inactivation of the
S446A
channels in which the C terminus of the
subunit was truncated are
due to some impairment of the interaction of the channels with the
cytoskeleton, one would expect that these effects will be occluded by
the DHCB treatment. Indeed, in oocytes of the same frogs the
inactivations of
S446A
and each one of its C-terminal
mutants were practically the same after DHCB treatment (Fig. 1B,
hatched bars). Thus, whereas the effect of DHCB was about 400% on
S446A
, it was more than 2-fold smaller (about 185%)
on either one of the mutants. The effects of DHCB on the
WT
channels (about 10%) and on its C-terminal
mutations were much smaller (Fig. 2B, hatched bars).
One possibility to account for the larger extent of inactivation of the
S446A
mutants (as compared with
S446A
itself) could be that the
-binding
capacities of the mutant polypeptides were larger. This possibility was
excluded in the concomitant biochemical analysis. SDS-PAGE analysis of
[35S]Met/Cys-labeled
and
polypeptides
coprecipitated by anti-
antibody showed that the
-binding
capacity of all the
S446A polypeptide species is
similar, namely, the same amount of
was coprecipitated per equal
amounts of any of the
polypeptides (Fig. 3). This
result is in accord with our previous data showing that under the
conditions of such an experiment the
-to-
ratio of the
coprecipitated polypeptides was the maximal possible, when >90% of
was saturated with
(21). The same SDS-PAGE analysis (Fig. 3)
shows that, as demonstrated before (16), the S446A
polypeptides
migrate as a 54-kDa polypeptide, which is the nonphosphorylated form,
whereas the WT
polypeptide migrates as a doublet of 54- and 57-kDa
polypeptides, the latter being the phosphorylated form. It should be
noted that the WT
polypeptide was basally phosphorylated to a large
extent, as is evident from the relative intensities of the 57- and
54-kDa bands.
Our preceding study (21) demonstrated that the inactivation of the
Kv1.1/Kv1.1 (
) channel is hampered by interaction of the
channel with microfilaments and that this interaction can be disrupted
to a significant extent by phosphorylation of a single C-terminal
residue (Ser-446) of the
subunit. Thus, in the present study, the
S446A
channel having the S446A
subunit, which, unlike the WT, is not basally phosphorylated in the oocyte, had a
substantially weaker inactivation (~10% of total current) than the
WT
channel (~60%). Consequently,
S446A
displayed a much stronger increase (by
~300%) in inactivation than
WT
(by ~10%) upon
treatment with DHCB, which disrupts the microfilaments (21). Therefore,
to look for the interaction between the
channel and the
cytoskeleton, rather than using the wild-type channel, we utilized the
S446A
mutant in which the channel-cytoskeleton interaction is functionally intact and which responds to any reduction in the strength of this interaction by a marked change in inactivation kinetics.
The interaction of the channel with microfilaments could occur
at sites on both the
and the
polypeptides. A highly probable
candidate was the consensus TDV sequence at the end of the C terminus,
recognized by the PDZ domain of the Dlg/PSD-95 family of proteins (for
reviews, see Refs. 8 and 24), thus providing cytoskeletal interaction
(9) and inducing clustering of Kv1.1 channels in COS7 cells (7). To
test this hypothesis, we studied the effects of truncation of the very
C terminus of
subunits, and of a point mutation in the TDV
sequence, on the extent of inactivation, both basal and following DHCB
treatment, of the
S446A
channels. One would expect
that if the interaction between the channel and the cytoskeleton was
disrupted by the mutations, the extent of inactivation would increase
and the enhancement of inactivation by DHCB would be weakened. Indeed,
the two different C-terminal mutations in the S446A
subunit (a
deletion of the last six amino acids and a replacement of the crucial
Thr-493 with alanine) significantly increased the basal extent of
inactivation of
S446A
and reduced the magnitude of
the inactivation enhancement by DHCB. As expected, under the conditions
of these experiments, in which the
WT
channels were
essentially detached from the cytoskeleton in their basal state in the
oocyte (as indicated by the large basal inactivation and small DHCB
effect), the same C-terminal mutations of the WT
subunit did not
increase the inactivation of the
WT
channels. This is
also in accord with the large extent of basal phosphorylation in the
oocytes used in these experiments (Fig. 3). In conclusion, the data
presented in this report corroborate the hypothesis that the very C
terminus of
interacts with the microfilaments.
The interaction of the very C terminus of the subunit with the
oocyte's cytoskeleton is via an endogenous protein in the oocyte,
possibly resembling protein members of the dlg/PSD-95 family. However,
truncation of all last six amino acids in the C-terminal tail was more
effective than substitution of Thr-493 in the TDV sequence with
alanine, a substitution that was shown to abolish the interaction of a
related
protein, Kv1.4, with PSD-95 (6). Thus, it seems that the
TDV motif in Kv1.1 is not the only determinant of
-subunit binding
to the postulated endogenous protein. In view of the demonstration that
the interaction of Kv1.1 with PSD-95 was much weaker than that of Kv1.4
in COS7 cells, and that Kv1.1 is missing a glutamate that precedes the
TDV sequence in Kv1.4 and was shown to be critical for the Kv1.4
interaction with PSD-95 (7), it seems plausible that Kv1.1 interacts
in vivo with a protein having a somewhat different
recognition sequence, yet to be identified. An alternative explanation
to the stronger interaction of Kv1.4 with PSD-95 could be that the
basal phosphorylation of this channel is weaker than that of Kv1.1 in
the cells tested.
It should be pointed out that the DHCB effect occluded the effects
caused by truncations of the C terminus of in
S446A
channels, much as it occluded (21) the effect
of Ser-446 phosphorylation of
in
WT
channels.
This suggests that disruption of microfilaments by DHCB causes maximal
enhancement of inactivation extent of the
channel experimentally
achievable, probably by a massive disruption of interactions between
the channels and the microfilaments. However, the fact that the effect
of DHCB was still prominent, though significantly reduced, in the C
terminus-truncated mutants suggests that there are additional sites of
interaction with the microfilaments on the
channel molecules
that modulate the inactivation.
The present study correlates the extent of inactivation of the
Kv1.1/Kv1.1 channel with its interactions with PSD-95-like proteins.
This is the first report that assigns a modulatory role to such
interactions. Such a functional interaction might be physiologically relevant at synaptic sites in different brain areas where PSD-95 family
members were shown to be concentrated and colocalized with Shaker-type
K+ channels including Kv1.1 (6).
We thank Dr. N. Dascal for helpful discussions.