(Received for publication, November 1, 1995; and in revised form, December 26, 1995)
From the
IsK is a 14.5-kDa type III membrane glycoprotein which induces
slowly activating K and Cl
currents
when expressed in Xenopus oocytes and HEK 293 cells. Recently,
mutagenesis experiments identified amino- and carboxyl-terminal domains
of IsK as critical for induction of Cl
and
K
currents, respectively. This hypothesis was tested
by examining effects of synthetic IsK hydrophilic peptides on untreated Xenopus oocytes. In agreement with IsK membrane topology, we
show here that peptides derived from carboxyl and amino termini are
sufficient to activate slow K
and Cl
channels whose biophysical and pharmacological characteristics
are similar to those exhibited by the native IsK protein. That data
provide further evidence that IsK is a regulatory subunit of
pre-existing silent channel complexes rather than a channel per
se.
The last few years have been an exciting time for our
understanding of the molecular structure and diversity of voltage-gated
K channels belonging to the Shaker-like
superfamily (for review, see (1, 2, 3, 4, 5) ).
Contrastingly, since its original cloning by expression in Xenopus oocytes, the nature of the IsK protein (or minK) remained a
mystery (for review, see (6) and (7) ). From a
structural point of view, IsK is an exception to the family of
K
channels. IsK is a 14.5-kDa type III glycoprotein
with one transmembrane segment which has no sequence homology with
other cloned functional channels(8) . It is a member of a
family of small bitopic membrane proteins which induce upon expression
in Xenopus oocytes slowly activating voltage-dependent
currents(6, 7, 9) . This family includes
phospholemman(10) , influenza virus M2 protein(11) ,
CHIF(12) , and Mat-8(13) . When expressed in Xenopus oocytes or in HEK 293 cells, the IsK protein evokes a
unique slowly activating, voltage-dependent
K
-selective current that closely resembles the slow
component I
of the cardiac delayed
rectifier(8, 14) . Two main hypotheses concerning the
nature of this protein were raised. The first was that IsK alone is
sufficient to form a voltage-gated K
channel, because
mutations in the transmembrane domain altered the gating of the
K
current expressed in Xenopus oocytes and
changed the relative permeabilities of NH
and Cs
versus K
(15, 16) . However, attempts to
express IsK as a K
channel in a variety of host cells
and after lipid bilayer reconstitution have failed(17) . The
second hypothesis was that IsK forms functional K
channels by association with an endogenous oocyte factor or with
pre-existing silent channels(18, 19, 20) .
Furthermore, IsK mutagenesis suggested that the amino-terminal domain
is critical for the induction of Cl
currents while
the carboxyl-terminal domain is critical for the activation of
K
channel activity(18) . These findings hinted
at the possibility that IsK is a regulatory subunit of heteromultimeric
channel complexes rather than a channel per se.
To test
this hypothesis, synthetic IsK hydrophilic peptides were applied to
untreated Xenopus oocytes. In agreement with IsK membrane
topology, we show here that internal or external application of IsK
peptides derived from the carboxyl- and amino-terminal domains are
sufficient to activate slow K or Cl
currents, respectively. The peptide-activated channels displayed
characteristics similar to those exhibited by the native IsK protein,
namely voltage dependence, activation kinetics, ion selectivity, and
pharmacology. Our data provide clear evidence for the nature of IsK as
a prototypic member of a family of short membrane transport proteins
with regulatory function.
Command pulse protocols, data
acquisition, and analyses were performed using pClamp software (Axon
Instruments, Foster City, CA) as described (23) . Oocytes were
perfused in OR2 standard solution, and all experiments were carried out
with the oocyte membrane held at -80 mV or -60 mV. Oocytes
were injected with full-length mouse IsK cRNA or full-length dog
phospholemman (10 ng/oocyte), and the resulting currents were recorded
2 days following cRNA injection. For intracellular peptide injection, a
volumetrically calibrated micropipette was used and peptide content in
20 nl was ejected by brief pressure pulses. The peptides were dissolved
in 20 mM HEPES (pH 7.4), 120 mM KCl and were briefly
sonicated before microinjection. To record depolarization-activated
currents, 20-s depolarizing pulses were applied from a holding
potential of -60 mV at 45-s intervals and 1 min after
microinjecting or superfusing the peptides. For C-27-activated
currents, the reversal potential determination was performed by
changing sequentially the perfusate to a series of OR2 solutions in
which Na was replaced by K
. For
extracellular peptide perfusion, the peptides were dissolved in OR2
superfusing solutions. To record hyperpolarization-activated currents,
5-s hyperpolarizing pulses were evoked from a holding potential of
-10 mV at 1-min intervals and 1 min after superfusing or
microinjecting the peptides. For N-34-activated currents, the reversal
potential determination was accomplished by changing sequentially the
perfusate to a series of OR2 solutions in which Cl
was replaced by MES. (
)Values from experiments with
multiple data points are expressed as mean ± S.E.
Mutagenesis experiments have shown that an intracellular
carboxyl-terminal domain of IsK is essential for the induction of the
slow K current in Xenopus oocytes(18) . A 27-mer hydrophilic peptide (C-27),
spanning a carboxyl-terminal region of the rat IsK sequence (positions
68-94) (22) and located immediately downstream from the
transmembrane segment, was synthesized and microinjected into Xenopus oocytes (Fig. 1). Within 30 s after C-27
peptide injection, membrane depolarization above a threshold of
-50 mV evoked a slowly activating voltage-dependent outward
current (Fig. 2, A and C). This current was
very similar to the slow K
current produced by the
native IsK. The depolarization-activated current could last for more
than 20 min after injection and then declined to 0 after 30 min.
Current amplitude was partly dependent on peptide concentration, being
activated above 20 µM C-27, but then rapidly saturating
above 100 µM C-27 (not shown). No currents could be
evoked, when internally applied C-27 was tested upon hyperpolarization
or when it was perfused in the external bath solution (n = 4, 3 batches). A 34-mer hydrophilic peptide(N-34),
spanning an amino-terminal region of the rat IsK sequence (position
10-43)(22) , did not evoke any current when injected into
the oocyte (at 200 µM; n = 5, 3 batches).
Other non IsK peptides such as P1, P2, and P3 (Fig. 1) were
ineffective at 200 µM (n = 5, 3 batches).
Figure 1: List of the rat IsK-derived and control peptides. Peptides were synthesized using the solid phase method and high performance liquid chromatography-purified as described under ``Experimental Procedures.''
Figure 2:
Expression of slow K currents in Xenopus oocytes microinjected with C-27 IsK
peptide. A, currents were evoked by 20-s depolarizing pulses
from a holding potential of -60 mV to + 60 mV in 20-mV
increments at 45-s intervals and 1 min after microinjecting 100
µM C-27. B, from a holding potential of -80
mV, tails of C-27-activated currents were recorded by a 20-s
depolarizing prepulse to +20 mV and followed by test potentials
between -30 mV and -130 mV in 20-mV decrements. C,
current-voltage relationships for water-injected (open
triangles), 10 ng of full-length IsK cRNA-injected (open
circles), and 100 µM C-27-injected (filled
circles) oocytes. The current amplitudes were measured at the end
of a 20-s depolarizing pulse from -50 mV to +50 mV. D, K
selectivity of C-27-induced currents
measured by tail current reversal potentials (open circles).
The straight line is the Nernst relationship for a perfectly
selective K
current. Points shown are the means
± S.E. of 4 independent experiments.
It was difficult to describe accurately the activation kinetics of
C-27-activated currents, since it did not reach steady-state even after
several minutes (at + 40 mV,
6-12
s, by fitting the 20-s pulse). Unlike deactivation kinetics, the
activation rates varied considerably among oocytes. Tail current
deactivation was faster than activation kinetics, requiring seconds for
full relaxation (Fig. 2B), and was fit as a
biexponential decay (at -80 mV repolarization,
= 0.27 ± 0.08 s and
=
1.80 ± 0.25 s; n = 7; 3 batches). The current
was selective for K
ions. The slope of the tail
current reversal potentials at various
[K
]
(substituted for
Na
) was 52.6 mV per decade, consistent with a
K
selective current (Fig. 2D; n = 4, in 2 batches). In Xenopus oocytes, voltage
steps to potentials above -20 mV usually give rise to transient
Ca
-activated outward Cl
currents in
OR2 standard recording solutions. To examine a possible interference of
outward Cl
conductance, we studied the C-27-activated
outward currents under Cl
-free recording solutions
(Cl
substituted with gluconate). Under these
conditions, Cl
influx was prevented and no
Ca
-activated Cl
currents could be
recorded in the presence of the Ca
ionophore A23187
(1 µM) at voltage steps from -20 mV up to 60 mV (not
shown). In gluconate recording solutions, intracellularly applied C-27
(100 µM) was able to evoke slowly activating
K
-selective currents upon step depolarization to 40 mV (Fig. 4A, control trace). Although the
activation was slightly faster than in Cl
-containing
solutions, the pharmacology was nearly identical with that found with
the expression of the native IsK protein. Fig. 4A shows
that barium (5 mM) and clofilium (100 µM) blocked
the C-27-induced K
current by 70 ± 9% and 60
± 10%, respectively (n = 5; 3 different
batches). Tetraethylammonium (30 mM) caused a 50 ± 11%
blockade of the current while lanthanum at 100 µM was
ineffective (n = 5, 3 batches). The lack of lanthanum
blockade is in contrast with a previous report (24) and
suggests that other domains of IsK are necessary to obtain an effective
inhibition. Similar results were obtained with aspartate or
methanesulfonate recording solutions (not shown).
Figure 4:
Pharmacology of C-27- and N-34-activated
currents. A, effects of clofilium (100 µM) and
Ba (5 mM) on K
currents
activated by 100 µM C-27, following a 20-s depolarizing
pulse to +40 mV (1 min after C-27 microinjection). B,
effects of Ba
(1 mM) and DIDS (1
mM) on Cl
currents activated by 50
µM N-34, following a 5-s hyperpolarizing pulse to
-150 mV (1 min after starting peptide superfusion; holding
potential -10 mV). C, ability of N-20 (200
µM) and N-13 (10 µM) to induce
hyperpolarization-activated currents as in B.
The role of the
amino-terminal domain of IsK in activating slow Cl currents was examined by applying extracellularly N-34 (Fig. 1) to untreated oocytes. Upon hyperpolarization, a slowly
developing inward current was evoked above a threshold of -90 mV (Fig. 3). This current was very similar to that induced by
phospholemman or by IsK (at high cRNA
concentration)(10, 18) . Since some batches of
untreated oocytes did express a similar slow inward current, we always
tested the effects of the peptides in oocytes that did not exhibit this
endogenous inward current. The N-34 action occurred within 15 s and
could be readily reversed upon washing out of the peptide, suggesting
that the peptide only weakly associates with the endogenous oocyte
channel. Like C-27 and above a threshold of 30 µM, current
amplitude was not strictly dependent on peptide concentration and
saturated above 100 µM (not shown). This lack of linear
concentration dependence suggests that the peptide binding to the
oocyte channels does not follow a simple bimolecular reaction.
N-34-induced current activated slowly with a sigmoidal waveform and
failed to reach steady state within 1 min, as described previously for
phospholemman(10) . Tail currents deactivated within less than
10 s at -10 mV, while the current relaxation process was much
slower at -40 mV (Fig. 3B). No currents were
evoked after depolarization or if N-34 (at 100 µM final
concentration) was injected intracellularly. C-27, P1, P2, and P3 (Fig. 1) were ineffective at 200 µM (n = 4, 3 batches).
Figure 3:
Expression of slow Cl currents in Xenopus oocytes perfused with N-34
amino-terminal IsK peptide. A, current records were evoked by
5-s hyperpolarizing pulses from a holding potential of -10 mV
between -90 mV to -170 mV in 20-mV decrements at 1-min
intervals and 1 min after superfusing 50 µM N-34. B, from a holding potential of -10 mV, tails of
N-34-induced currents were studied using a 5-s hyperpolarizing prepulse
to -150 mV and followed by test potentials between -40 mV
and +20 mV in 10-mV increments at 1-min intervals. C,
current-voltage relationships for water-injected (open
triangles), 10 ng of full-length phospholemman cRNA-injected (open circles), and 50 µM N-34-perfused (filled circles) oocytes. The current amplitudes were measured
at the end of a 5-s hyperpolarizing pulses from 0 mV to -200 mV. D, Cl
selectivity of N-34-induced currents
measured by the tail current reversal potentials (open
circles). The straight line is the Nernst relationship
for a perfectly selective Cl
current with a
[Cl
]
= 65
mM. Points shown are the means ± S.E. of 3 independent
experiments.
To determine the ionic selectivity
underlying the N-34-induced inward current, we measured the dependence
of the tail current reversal potential on
[Cl]
(Fig. 3D).
As expected for Cl
channels, the currents reversed
direction at about -25 mV in standard solutions, and the reversal
potential shifted to more positive values, when
[Cl
]
decreased (Fig. 3D). However, the deviation of the curve from the
Nernst relation, especially at low
[Cl
]
, suggests that other
conductances may also be involved. This feature is reminiscent of that
found for phospholemman-induced slow inward currents (10) and
for slow endogenous oocyte channels
I
In order to narrow down more precisely the active domain of the IsK amino-terminal, we looked at the effects of two externally applied peptides (Fig. 1). N-20, which overlaps with the proximal portion of N-34, was inactive (up to 200 µM; n = 3, 2 batches) ( Fig. 1and Fig. 4C). By contrast, N-13 (amino acids 31-43) was active and reached its maximal effect at 10 µM ( Fig. 1and Fig. 4C; n = 3, 2 batches). Thus, this short domain suffices by itself to act on the channel complex.
In this report we have shown that intracellular or
extracellular application of specific hydrophilic IsK peptides was
sufficient to mimic the native IsK induction of slow K and Cl
currents(6, 7, 8, 18) . It is
clear that these peptides could not form by themselves ion-conducting
pores, since they do not interact with the membrane and their action is
not irreversible. The peptide-induced channel activity is specific
since non-IsK peptides were ineffective. The specificity of the peptide
interaction is further evidenced by the ability of intracellular
carboxyl terminus and extracellular amino terminus peptides to
exclusively evoke K
or Cl
currents,
respectively. This is in agreement with the IsK membrane topology. Our
data strongly support the notion that IsK must associate with some
endogenous oocyte component to form a functional channel
complex(18, 19, 20) . However, mutations in
the transmembrane domain of IsK were found to alter channel
selectivity, open channel block, and gating
kinetics(15, 16) . To explain this apparent
controversy, we suggest that IsK, acting as a regulatory subunit, has
some contribution in defining the K
pore properties
and in modulating channel gating. This is actually the case with the
1 subunit of voltage-dependent Na
channels (same
membrane topology as IsK) and voltage-dependent Ca
channels which alter the voltage dependence of channel
gating(26) .
It has been suggested recently that IsK might
become functional by interacting with either a rare lipid, a
cytoskeletal protein, or a channel protein subunit(19) .
Regarding our data, we favor the latter proposal for two main reasons.
First, it is energetically unfavorable for a hydrophilic peptide to
associate with a lipid to form a functional channel. Second, it seems
unlikely that an extracellularly applied peptide such as N-34 will
interact with an intracellular cytoskeletal component to produce a
current. Naturally, it does not exclude that the whole channel complex
could be linked to the cytoskeleton. Thus, the reasonable explanation
is that the IsK peptides interact with endogenous oocyte channels to
activate them. In such a model, IsK may act at least in one of the two
ways. 1) It could function as an activator or a regulatory subunit
which activates pre-existing silent channels by direct protein-protein
interactions. 2) Alternatively, IsK could act by recruiting inactive
channels and functions as a chaperone that facilitates assembly of
multimeric channel complexes. However, the relatively fast peptide
action together with the ability of externally applied N-34 or N-13 to
evoke slow Cl currents are not compatible with this
view.
The oligomeric nature of the IsK channel complex remains
unknown; however, recent studies suggested that it could be made of
just two IsK monomers associated with as yet unidentified non-IsK
subunits(20) . The very slow gating kinetics of IsK suggest
that it could activate by a unique mechanism. A model in which IsK
channels activate by voltage-dependent subunit aggregation has been
proposed (27) . Cross-linking or Hg-induced
chelation of IsK subunits were found to hold the channel complex in an
open conducting state(27, 28) . This mechanism of
subunit aggregation also accommodates our view of a heteromultimeric
subunit oligomerization process.
It is clear that the slow
K and Cl
currents are not
specifically and exclusively induced by the IsK protein in Xenopus oocytes. Other structurally similar small bitopic membrane
proteins like CHIF, Mat-8, or phospholemman are also able to activate
these slowly activating
currents(10, 12, 13) . For example, IsK and
CHIF evoke slow K
currents which fail to reach steady
state within tens of seconds and are sensitive to block by
Ba
and clofilium. Since these proteins share no
sequence similarity, it suggests that the specificity requirements for
binding to the endogenous channel complex are relatively low. The
conformation that the peptides adopt upon binding is unknown; however,
the various peptidic motifs must share some yet undefined structural or
conformational similarities. The transient character of the peptide
action suggests that the transmembrane domain and may be other regions
such as the extreme amino terminus may help in stabilizing the physical
interaction between IsK and other subunits of the channel complex,
independently of the gating process.
In conclusion, our findings provide further evidence for the nature of IsK as a member of a family of short bitopic membrane proteins which are capable of activating endogenous and otherwise silent ion channels. It is now crucial to investigate which physiological roles subserve IsK-channel protein interactions in epithelia, T lymphocytes, or cardiac cells and what are the molecular structures of these interacting channel proteins.