(Received for publication, October 23, 1996, and in revised form, November 14, 1996)
From the Departments of Pediatrics and Cellular and Molecular Physiology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812
MinK is a transmembrane protein of 130 amino acids found in the kidney, heart, and vestibular system of mammals. Its expression in Xenopus laevis oocytes induces a voltage-dependent potassium current similar to that seen in vivo. Indirect evidence has fueled speculation that function requires association of MinK and another protein endogenous to oocytes and native tissues. In this report, we show that direct covalent modification of an oocyte membrane protein alters properties of the MinK ion conduction pore; modified channels exhibit decreased potassium conduction and increased permeability to sodium and cesium. The modifying reagents, two membrane-impermeant, sulfhydryl-specific methanethiosulfonate derivatives, react only from the extracellular solution at rates that are determined by the conformational state of the channel. These findings indicate that MinK is intimately associated with an oocyte protein whose exposure to the external solution changes during channel gating and which acts with MinK to establish ion conduction pore function.
Ion channel proteins form transmembrane aqueous pores that open and close in response to specific stimuli and thereby mediate cellular electrical activity. Voltage-gated K+ channels govern the activity of cyclically excitable tissues (like nerves and muscles) by acting to repolarize cells after stimulation. Originally isolated from rat kidney based on its ability to induce voltage-activated K+ currents in Xenopus oocytes (1), MinK enjoys wide tissue distribution in mammals (2). In oocytes, MinK currents exhibit many characteristics of classical voltage-dependent, K+-selective channels (3, 4) and show notable similarities to the delayed rectifier K+ currents present in cardiac myocytes (2, 5-7) and vestibular dark cells (8); this has led to the hypothesis that MinK underlies these currents in vivo.
Because MinK bears no sequence homology to other K+ channel
proteins, uncertainty has surrounded its role in channel function. Early confusion that MinK could regulate Cl channels (9)
has proven to be an artifact of over-expression (10, 11) and given way
to the sentiment that MinK is a structural protein intrinsic to channel
formation (4). Thus, point mutations have identified MinK residues that
modify almost all channel functions including activation by voltage
(12, 13), regulation by protein kinase C (14, 15), ionic selectivity
among monovalent cations (4), and sensitivity to inhibition by open
channel pore blockers (4, 16). Recently, we showed that a domain of
MinK lines the external portion of the ion conduction pore (16). When
mutated to cysteine, sites in this domain render channels susceptible to covalent block by methanethiosulfonate ethylsulfonate
(MTSES
)1 and alter its
reversible inhibition by tetraethylammonium (TEA). That TEA blockade is
sensitive to the concentration of permeant ions inside the cell (as
well as transmembrane voltage) and that MTSES
and TEA
compete for overlapping MinK binding sites argue that both bind to the
external end of the MinK conduction pore, ~15% of the way through
the potential drop across the membrane.
The subunit composition of MinK channels has also been a matter of controversy (13, 17). With the exception of MinK, cloned K+ channel subunits possess one or two highly conserved, pore-forming P domains (18, 19), while voltage-gated Ca2+ and Na+ channel subunits contain four P domains (20). In those channels, the ion conduction pathway is formed by four P domains supplied by four, two, or one subunit, respectively (21).2 MinK has no P domains by which to predict its subunit stoichiometry. We have supported a heteromultimeric model containing two MinK monomers, based on the effects of co-expression of MinK and MinK mutants (13), and a required non-MinK subunit that oocytes provide but only in a limited amount since isochronal currents saturate despite increasing levels of MinK protein on the oocyte surface (13, 16). A role for a non-MinK subunit has also been proposed by Blumenthal and Kaczmarek (22) based on similar findings and by Lesage et al. (23) based on their study of three cell lines that fail to show currents despite MinK surface expression. Others have speculated that MinK may associate with HERG K+ channel subunits based on inhibition of IKr currents in a cardiac cell line treated with MinK antisense oligonucleotides (24).
In this report, we show that K+ channels induced in oocytes by a cysteine-free mutant of MinK are sensitive to two sulfhydryl-specific reagents that covalently modify free cysteine residues exposed to aqueous solvent. Modification alters MinK ion conduction and ion selectivity, two fundamental properties of the channel pore. Channels are sensitive to modification only from the extracellular solution, and the rate of reaction depends on the channel conformational state. These results indicate that a cysteine-bearing protein endogenous to oocytes is intimately associated with the channel gating and conduction apparatus and suggest it may contribute directly to the MinK pore.
Mutants of rat MinK were made in pSD (16) and cRNA (2 ng)
injected into oocytes. Whole cell currents were measured after 2 days
by a two-electrode voltage clamp with constant perfusion at 22 °C
(13). Data were filtered at 100 Hz, sampled at 1 kHz, and leak
correction was performed off-line (3, 4). Bath solution was ND-96 (in
mM): 96 NaCl, 2 KCl, 1 MgCl2, 0.3 CaCl2, and 5 HEPES, pH 7.6) or solutions in which NaCl and
KCl were replaced by 98 mM test cation as chloride salt.
MTS solutions were made from powder (Toronto Research, Ontario) by
substitution for NaCl in ND-96 and used within 15 min. MTS derivatives
hydrolyze in aqueous solution (16, 25). The half-life in ND-96 at
22 °C for 5 mM MTSES was ~55 min; for
2.5 mM MTSEA+, it was ~ 40 min; and for
10 mM MTSET+, it was ~20 min; hydrolyzed 2.5 mM MTSEA+ was prepared by incubation at
22 °C overnight.
A panel of water-soluble,
membrane-impermeant, thiol-specific methanethiosulfonate (MTS)
derivatives developed by Akabas, Karlin and co-workers (25, 26) has
gained wide use for evaluation of sites in membrane proteins exposed to
aqueous solution (27). These reagents are over 2,500 times more soluble
in water than n-octanol and form mixed disulfides with free
sulfhydryls accessible to aqueous solvent; in proteins, a covalent bond
forms between a portion of the reagent
(-SCH2CH2X) and exposed cysteine
side chains. In previous work, we found that a negatively charged MTS derivative, MTSES, did not block wild-type MinK channels
in oocytes (Fig. 1) but did block MinK mutants bearing
cysteine at positions 44, 45, or 47 (16).
On the other hand, wild-type MinK is blocked by two positively charged MTS derivatives, MTS-ethylammonium (MTSEA+) and MTS-ethyltrimethylammonium (MTSET+) (Fig. 1). The single native cysteine in MinK at position 107 is thought to be intracellular and protected from reagents in the external solution. As expected, mutation of this cysteine to alanine (C107A-MinK) does not relieve block by the two cationic MTS reagents (Fig. 1). This suggests that a non-MinK, cysteine-bearing protein is modified, rather than MinK, to alter channel function. MTS derivatives show marked specificity for free sulfhydryls (25, 28) and alter ion conduction through both anion- and cation-selective channels with free cysteines in their pores (16, 26, 29-32). Thus, a simple model to explain block of MinK by MTSEA+ and MTSET+ is that a protein native to oocytes associates with MinK and exposes a free cysteine close to the MinK channel pore. We sought to support or refute this idea by studying in detail the effects of MTSEA+ on C107A-MinK.
Block of C107A-MinK by MTSEA+ exhibits characteristics
expected for covalent modification, it is not reversed upon washout and
does not occur with hydrolyzed reagent. Exposure of oocytes expressing
C107A-MinK to 2.5 mM MTSEA+ for 5 min or more
decreases currents by 87 ± 5% (mean ± S.E., 17 oocytes).
Higher concentrations of MTSEA+ (up to 25 mM),
longer periods of exposure (up to 20 min), and washout periods up to 30 min do not alter the magnitude of irreversible blockade. This type of
stable partial blockade is commonly observed after treatment of
channels with MTSEA+ (16, 26, 29-32) and has been shown to
reflect a decrease in single-channel conductance in two cases (31, 32).
Only weak reversible inhibition is observed when C107A-MinK expressing
oocytes are treated with 2.5 mM hydrolyzed
MTSEA+, 5 ± 2% (mean ± S.E., 6 oocytes).
Inhibition by MTSEA+ is unexpectedly specific; irreversible
blockade is not observed when C107A-MinK is treated to a panoply of
thiol-specific reagents (for 8 min or more) including 30 mM
iodoacetic acid, 30 mM iodoacetamide, 1 mM
3-(N-maleimidopropionyl)-biocytin, 1 mM
N-ethylmaleimide, 1 mM
1-biotinamido-4-(4-[maleimidomethyl]cyclohexane-carboxamido)butane, 0.4 mM
N-[6-(biotinamido)hexyl]-3
-(2
-pyridyldithio)propionamide, 2 mM fluorescein mercuric acetate, or 50 mM
thimerosal.
That both MTSEA+ and MTSET+ (with a fixed positive charge) block irreversibly from the extracellular solution suggests the modification site is accessible to the bath solution and not buried in a hydrophobic portion of the channel or membrane. This is supported by the observation that intracellular microinjection of 23 nl of 50 mM MTSEA+, yielding an intracellular concentration of ~2.5 mM MTSEA+, produces a current reduction of only 6 ± 5% (mean ± S.E., 4 oocytes measured at 10 and 30 min after injection).
MTSEA+ Block of C107A-MinK Is Faster When Channels Are ClosedThe kinetics of MinK blockade by MTS derivatives can be
studied by abrupt exposure of oocytes expressing channels to the
reagents (16). To test whether MTSEA+ modification is
sensitive to channel conformation, we measured the rate of block of
C107A-MinK currents with duty cycles of varying durations at the test
voltage (0 mV) and holding voltage (80 mV) (Fig. 2).
When oocytes are exposed to 2.5 mM MTSEA+
during cycles with 3-s test pulses and 10-s holding intervals, blocking
kinetics follow a roughly exponential relaxation with an apparent time
constant (
) of 112 ± 12 s (mean ± S.E., 8 oocytes). When the fraction of time spent at the holding voltage is decreased, by
increasing the test pulse to 10 s and maintaining a 10-s holding interval, the rate of channel block is slower,
= 185 ± 25 s (mean ± S.E., 5 oocytes). The rate of block slows
further when the 10-s test pulse was maintained, but the interpulse
holding voltage interval is reduced to 3 s,
= 262 ± 32 s (mean ± S.E., 5 oocytes). While the magnitude of block
achieved in all 3 protocols is unchanged, the rate of block slows as
the fraction of time spent at the test potential increases. This
demonstrates that MTSEA+ reacts more readily at voltages
that favor the closed channel state and suggests a close association of
MinK gating structures and the binding site for MTSEA+
since its exposure changes as channels move between closed and open
states. A similar state-dependent enhancement of binding site exposure is observed when Shaker K+ channels carry a
cysteine in their external pore region and move from open to an
inactive conformation (33, 34).
The effect of voltage on block kinetics was evaluated by employing a
constant duty cycle (3-s test pulse, 10-s holding interval) and
comparing test pulse voltages of 0 and +40 mV with a holding voltage of
80 mV. The rate of MTSEA+ block is slower at the more
positive potential,
= 160 ± 14 s (mean ± S.E., 4 oocytes), although the final magnitude of block is unchanged. This is
consistent with the predicted effect of voltage on block by a
positively charged agent that moves into the ion conduction pore and,
thus, experiences the transmembrane electric field (4, 35). However,
the effect of voltage may be a manifestation of
state-dependent block and a slowly activating channel,
since the more positive potential moves channels out of the
MTSEA+ reactive state more rapidly. Thus, despite this
voltage dependence, the MTSEA+ site may be superficial to
the transmembrane electric field and the influence of voltage secondary
to state-dependent exposure.
While the kinetics of MTSEA+ blockade are sensitive to channel state, modification does not lead to inhibition of current as a result of slowed channel opening or speeded channel closure (Table I). Although the activation of MinK channels are complex (36, 37), a qualitative approximation of activation kinetics can be achieved by comparing the amplitude of currents at 2 and 10 s while deactivation kinetics are well described by the sum of two exponentials (13). In fact, channels activate slightly more rapidly after MTSEA+ modification than before and close slightly more slowly. If this were the only effect of MTSEA+ on MinK, treatment would be expected to augment, rather than diminish, currents. This indicates that another mechanism underlies inhibition, perhaps direct pore occlusion, as seen with MTS reagents in other K+ channels (31, 32).
|
To test whether MTSEA+
modification alters properties associated with the MinK pore, ion
selectivity can be assessed by tail current reversal potential
measurements (Fig. 3). Oocytes are held at 80 mV,
pulsed to +20 mV to open the channels, and then repolarized to various
test potentials; the initial slopes of tail currents are examined to
determine reversal potential (Vrev). Unmodified
C107A-MinK channels, like wild-type MinK (4), exhibit nearly ideal
selectivity for K+ over Na+, showing a change
of 55 ± 2 mV in Vrev with a 10-fold change in bath K+ concentration (Table I), close to the 58-mV
shift predicted by the Nernst relationship. On the other hand,
MTSEA+-modified channels shift their reversal potential
only 38 ± 2 mV (Table I). This indicates that ion selectivity of
modified channels has been altered.
To investigate how selectivity is altered, the fine ionic
discrimination among monovalent K+ ion analogs by
unmodified and modified channels can be compared. Tail current reversal
potentials are analyzed in terms of the Goldman-Hodgkin and Katz
relation with each test ion as the only monovalent cation in the bath
solution (Table II) (4). These "pseudo-biionic"
conditions allow a comparison of the relative permeability of the test
ion and K+ (the predominant permeant ion inside the cell)
before and after MTSEA+ exposure. MTSEA+
modification does not alter the relative permeability series for
C107A-MinK channels (K+ > Rb+ > Cs+ > Na+ Li+,
NMDG+) or their ability to exclude Li+ and
NMDG+ (Table II). However, modification does increase the
relative permeability of channels for both Cs+ and
Na+ by at least 2-3-fold (Fig. 3 and Table II).
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An increase in Na+ permeability through modified MinK channels should decrease the net outward current seen with depolarization since inward Na+ flux will now offset outward K+ current. This mechanism underlies the loss of K+ currents in granule cells of weaver mice and results from a mutation in the pore of their GIRK2 K+ channels (38, 39). However, increased Na+ permeability is insufficient to explain the majority of the observed current reduction in MTSEA+-modified MinK channels; permeability for K+ remains 100-fold greater than for Na+, and tail currents do not show augmented Na+ conductance through modified channels (Fig. 3). Of note, increased Na+ permeability does not develop in a linear relationship with the appearance of MinK channel blockade; no change in Na+ permeability is seen until more than 70% of the current is inhibited when reversal potentials are measured in 98 mM Na+ solution (n = 16 oocytes). That initial modification events lead only to channel blockade while later events result in changes in ion selectivity argues that more than one reactive cysteine is modified in each channel (on one or more channel subunits).
Permeability changes cannot be attributed to complete blockade of MinK channels and the exposure of less-selective ion channels endogenous to oocytes. Such endogenous currents are not seen in control cells. Moreover, residual unblocked currents exhibit characteristics of MinK channels including slow, voltage-dependent activation, rapid deactivation, sensitivity to barium, and voltage-dependent blockade by TEA (Table I). Partial blockade of MinK by MTSEA+ is like the effect of this agent on other K+ channels (31, 32, 34).
Block by TEA or MTSESPreviously, we found that TEA binds in
the external pore of the MinK channel, and its presence in the bath
solution slows the rate of reaction of negatively charged
MTSES with a A45C/C107A-MinK, a mutant with a cysteine in
position 45; this suggests the sites for TEA and MTSES
overlap (16). Conversely, TEA and MTSEA+ do not appear to
share overlapping binding sites since the rate of block of C107A-MinK
by 2.5 mM MTSEA+ is not altered by the presence
of 75 mM TEA,
= 111 ± 16 s (mean ± S.E., 5 oocytes). Consistent with this conclusion, the equilibrium inhibition constant (Ki) for TEA blockade of
C107A-MinK is the same before and after MTSEA+ modification
(Table I). Sites for MTSEA+ and MTSES
are
also non-overlapping. After complete reaction of A45C/C107A-MinK channels with MTSES
, the magnitude and rate of subsequent
MTSEA+ modification is unchanged,
= 104 ± 9 s (mean ± S.E., 6 oocytes).
In the present study, we demonstrate that two sulfhydryl-specific reagents, MTSEA+ and MTSET+, alter the function of cysteine-free MinK channels in oocytes. The reagents react from the extracellular solution in a state-dependent fashion, modifying channels more rapidly when they are closed. Modification acts to change two attributes of MinK pore function. First, ion conduction is inhibited, apparently by a blocking mechanism since channel gating remains largely unchanged. Second, the channels' selectivity among monovalent cations is altered. That modification leads first to blockade and subsequently to changes in ion permeability suggests that more than one site is modified. Based on these results and other recent work (13, 16), we propose that MinK channels are formed through the intimate association of two MinK monomers and one or more copies of an oocyte membrane protein. Both MinK and the oocyte protein are exposed to the external solution, both change their local environment during channel gating, and both contribute to ion conduction pore function.
Negatively charged MTSES, while somewhat smaller than
MTSET+, has no effect on C107A-MinK. This suggests that the
MTSET+ site, if in the channel pore, is beyond the position
that selects against permeation by negative ions. Like point mutation
of residues in the pore-forming P domains of other K+
channels (38-40), MTSEA+ modification alters selectivity
filter function. While it is tempting to speculate that modification
occurs in proximity to the MinK channel selectivity filter (and,
further, that the reactive site is near a P-like domain on the oocyte
protein), it is important to emphasize that our results do not provide
information about the mechanism by which ion selectivity is altered or
show that modification has a local effect on ion permeation. While the
findings are consistent with the idea that MTSEA+ binds in
the channel pore, such a conclusion cannot be made from studies of ion
permeation alone. The findings do allow us to answer the question
motivating this study, one or more copies of a cysteine-bearing oocyte
protein are intimately associated with each MinK channel.
The wide diversity of K+ channel functions observed in vivo reflect the many genes encoding K+ channel subunits, production of splice variants, and the heteromultimeric association of channel subunits with distinctive properties (20). While some channels are functional as homomeric complexes, many are functional only when different subunit species co-assemble (41, 42). One such K+ channel, GIRK1, whose natural partner is the cardiac inward rectifier subunit CIR (41), is able to function in oocytes only because it can assemble with a channel subunit endogenous to oocytes (XIR) to form functional channel complexes (43). MinK channels have been speculated to require co-assembly of MinK and another protein endogenous to oocytes, cardiocytes, or vestibular dark cells (13, 16, 22, 23). We show here that functional MinK channels are, indeed, heteromultimeric.
Consistent with our findings and conclusions, two reports now indicate that MinK protein can form functional heteromultimeric channels with KvLQT1, a single P domain K+ channel subunit present in human heart (Sanguinetti, M. C., Curran, M. E., Zou, A., Shen, J., Spector, P. S., Atkinson, D. L., and Keating, M. T. (1996) Nature 384, 80-83 and Barhanin, J., Lesage, F., Guillemare, E., Fink, M., Lazdunski, M., and Romey, G. (1996) Nature 384, 78-80). Sanguinetti et al. also reveal the partial predicted sequence of a Xenopus laevis homolog (XKvLQT1) which may prove to be the cysteine-bearing protein endogenous to oocytes whose association with MinK we study here.