Correspondence to: R.S. Kass, Department of Pharmacology, College Columbia University, 630 West 168 Street, New York, NY 10032. Fax:(212) 342-2703 E-mail:rsk20{at}columbia.edu.
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Abstract |
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IKs, a slowly activating delayed rectifier K+ current through channels formed by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contributes to the control of the cardiac action potential duration. Coassembly of the two subunits is essential in producing the characteristic and physiologically critical kinetics of assembled channels, but it is not yet clear where or how these subunits interact. Previous investigations of external access to the KCNE1 protein in assembled IKs channels relied on occlusion of the pore by extracellular application of TEA+, despite the very low TEA+ sensitivity (estimated EC50 > 100 mM) of channels encoded by coassembly of wild-type KCNQ1 with the wild type (WT) or a series of cysteine-mutated KCNE1 constructs. We have engineered a high affinity TEA+ binding site into the h-KCNQ1 channel by either a single (V319Y) or double (K318I, V319Y) mutation, and retested it for pore-delimited access to specific sites on coassembled KCNE1 subunits. Coexpression of either KCNQ1 construct with WT KCNE1 in Chinese hamster ovary cells does not alter the TEA+ sensitivity of the homomeric channels (IC50 0.4 mM [TEA+]out), providing evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Coexpression of a cysteine-substituted KCNE1 (F54C) with V319Y significantly increases the sensitivity of channels to external Cd2+, but neither the extent of nor the kinetics of the onset of (or the recovery from) Cd2+ block was affected by [TEA+]o at 10x the IC50 for channel block. These data strongly suggest that access of Cd2+ to the cysteine-mutated site on KCNE1 is independent of pore occlusion caused by TEA+ binding to the outer region of the KCNE1/V319Y pore, and that KCNE1 does not reside within the pore region of the assembled channels.
Key Words: heart potassium channels, pore, cysteine substitution, LQT-1, subunit assembly
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INTRODUCTION |
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IKs, a slowly activating delayed rectifier K+ current through channels formed by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contributes to the control of the duration of cardiac action potentials (
To determine whether sites of the KCNE1 protein are exposed within the IKs pore,
Here, we have reinvestigated pore-delimited access of the thiol-reactive reagent (Cd2+) to specific cysteine-mutated sites (F54C and G55C) on the h-KCNE1 protein by coexpressing these constructs with highly TEA+-sensitive mutant h-KCNQ1 channels (K318I + V319Y; V319Y) in transiently transfected Chinese hamster ovary (CHO)1 cells. Externally applied TEA+ rapidly, reversibly, and potently blocked channels consisting of these mutants alone or coassembled with WT and mutant KCNE1. We tested for, but did not find evidence of, restrictions of Cd2+ access to F54C or G55C when this KCNE1 mutant was coexpressed with the TEA+ sensitive mutants. Our data strongly suggest that KCNE1 does not reside within the pore region and that any effects of KCNE1 assembly are likely to be due to interactions between KCNE1 and KCNQ1 that reside elsewhere, perhaps with the S4 segments themselves.
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MATERIALS AND METHODS |
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Molecular Biology
The human cDNA clone KCNQ1 was a gift from Dr. M. Keating (Department of Human Genetics, University of Utah, Salt Lake City, UT) and subcloned into the mammalian expression vector pCDNA3.1 (Invitrogen). Mutations of both KCNQ1 and KCNE1 were performed by plaque-forming unit-based mutagenesis (QuickChangeTM site-directed mutagenesis kit; Strategene), and the mutant gene fragments were inserted into translationally silent restriction sites. All sequences were performed by the chain termination method in the DNA Sequencing Facility at Columbia University. The basic protocol uses Chinese hamster ovary (CHO) cells that were cultured in Ham's F12 medium and transiently transfected using Lipofectamine with Lipofectamine-PLUS reagents (Life Technologies) as previously reported (
Electrophysiology and Data Analysis
Currents were recorded using the whole-cell patch-clamp technique ( when filled with the following internal solution (in mM): 110 potassium aspartate, 5 ATP-K2, 11 EGTA, 10 HEPES, 1 CaCl2, and 1 MgCl2, pH 7.3. Series resistance was 3.58 M
and was electrically compensated.
Transmembrane currents were obtained using Axopatch 200A amplifiers (Axon Instruments) with 100-M headstages, low-pass filtered at 2 kHz, digitized at 0.5 kHz, and sampled online to a computer hard disk. Activation (isochronal) of IKs was studied by applying 2-s depolarizing pulses in 20-mV increments from -40 mV to approximately -60 mV of holding potentials. In most of the experiments, test potentials were applied to 60 mV at 0.33 Hz. Activation was measured as the time-dependent current during test pulses or as deactivating current tails after test pulses. Most voltage protocols were designed to terminate within
12 min to minimize rundown of the currents. pCLAMP software (version 8.0; Axon Instruments) was used both to generate the voltage-clamp protocols and to acquire data. Graphical and statistical data analysis was carried out using Origin 6.0 software (Microcal). Statistical significance was assessed with t test for simple comparisons: differences at P < 0.05 were considered to be significant.
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RESULTS |
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External TEA+ Block of Wild-type and Mutant IKs Channels
Because it is generally assumed that KCNQ1 encodes the pore forming subunit of functional IKs channels (
We next investigated the effects of these KCNQ1 mutations on gating and sensitivity to [TEA+]o with and without coexpression of KCNE1. In the absence of KCNE1, wild-type (WT) KCNQ1 channels were not completely blocked by >50 mM TEA+ as previously reported (
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Mutant heteromultimeric channels retain the TEA+ sensitivity of the homomultimer (Fig 1 C), as is the case for WT KCNQ1 subunits. This result provides evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Furthermore, since we measured the same TEA+ sensitivity for KCNE1 expressed with either the single or double KCNQ1 mutant, we used the V319Y KCNQ1 construct as a tool for highly TEA+-sensitive IKs channels in the remaining experiments.
External Cd2+ Sensitivity of WT and Cysteine-mutated KCNE1 Constructs
We next coexpressed the TEA+-sensitive KCNQ1 construct (V319Y) with the cysteine-mutated KCNE1, and determined the external Cd2+ sensitivity of the KCNQ1KCNE1 channel complex. Exposure of V319Y/ WT KCNE1 channels to 2 mM Cd2+ reversibly blocked the expressed current by <10% (30-s exposure). This result suggests a subtle involvement of endogenous cysteines, which causes a background block of expressed channels. We next sought to determine whether or not engineering a cysteine into KCNE1 increases the Cd2+ sensitivity of the encoded channels.
We tested the Cd2+ sensitivity of WT KCNQ1 coexpressed with mutant (F54C) KCNE1 (Fig 2 B), and found the mutation significantly increased 2 mM Cd2+ block approximately threefold as shown previously (
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Because
Rapid Kinetics of TEA+ Block
Our rapid application system (time constant >20 ms) allowed us to investigate the effects of TEA+ during 2-s depolarizing pulses. As shown in Fig 3, exposure of 10 mM TEA+ to V319Y IKs completely blocked the expressed current within 500 ms, and the block can be washed out within 200 ms. Because of the slow kinetics of the channels, both wash in and wash out could be measured during a single depolarizing pulse. The rapid and reversible block by TEA+ is consistent with interactions at the mutated site in KCNQ1 (position 319), which is equivalent to Shaker 449T (
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Onset of Cd2+ Block in the Absence and Presence of TEA+
Thus, we next tested for the possibility that the interaction of Cd2+ with the Cys at position 54 of KCNE1 is affected by TEA+ block of the channels. We carried out two sets of experiments to test for this possibility. First, we measured the time course of the onset and recovery of Cd2+ block in the absence and presence of a saturating TEA+ concentration. If the pore of the IKs channel is occluded completely by high concentration of TEA+, and if Cys54 was located within the pore, then we reasoned that accessibility of Cd2+ to Cys54 would be restricted by TEA+ block.
The protocol we chose took advantage of the application of TEA+ and Cd2+ via the rapid solution change system illustrated in Fig 3 and applied in Fig 4 and Fig 5. For these experiments, the same concentration of Cd2+ application was applied in the absence or presence of TEA+ in the same cell. In the absence of TEA+, we measured the onset of and recovery from Cd2+ block of the channels. In the absence of TEA+, the application of Cd2+ resulted in approximately a 30% reduction in the current (also see Fig 2 D), which could be completely reversed with wash out (Fig 4). We repeated the experiment, but in this case first applied TEA+ at a sufficiently high concentration (10 mM) to completely block the channels. It was possible to assay Cd2+ block of the channel that had occurred in the presence of total channel block by TEA+ by quickly washing out TEA+ and measuring the amplitude of the remaining current (Fig 4A and Fig C). Washout of TEA+ revealed Cd2+-blocked channel activity remarkably similar to that obtained in the absence of TEA+ (Fig 4, compare B and C). This and other similar experiments are summarized in Fig 5, which confirms the observation that the magnitude Cd2+ inhibition of the V319Y/F54C channel is significantly different from inhibition of V319Y/WT KCNE1 channels, but is not affected by the presence of 10 mM TEA+. These data suggest that pore occlusion by TEA+ does not prevent Cd2+-access to the Cd2+ sensitive site on KCNE1 (position 54).
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Influence of TEA+ on the Kinetics of Cd2+ Block
Binding of TEA+ and Cd2+ to their respective sites of interaction is a dynamic process and possible effects of TEA+ on Cd2+ binding will be determined by the on and off rates of the two ions. Assuming pore-limited access of Cd2+ to the F54C KCNE1 site, a simple model for Cd2+ binding is shown in Scheme 1. In the simplest case, because of rapid TEA+ kinetics, TEA+ is in equilibrium (within dotted square in Scheme 1), and Cd2+ is assumed to bind only to the fraction of channels that are not blocked by TEA+ (fUB). This fraction of channels is defined as:
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(1) |
where k+1(TEA) and k-1(TEA) represent the apparent onset rate constant (s-1M-1) and the apparent off rate constant for TEA+ (s-1), respectively. The kinetics of Cd2+ block of the channel can be calculated by considering the interaction of Cd2+ with channels that are not blocked by TEA+, or fUB derived above. The equation for the time constant () of Cd2+ block (unblock) of channels in the presence of TEA+ will be:
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(2) |
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(3) |
where is the time constant in seconds, L is the concentration of Cd2+ in moles, k1 is the apparent onset rate constant (s-1M-1), and k-1 is the apparent off rate constant for Cd2+ (s-1). To test this prediction, experimental determination of the parameters in Equation 3 is needed. The data in Fig 1 can be used to estimate fUB as a function of [TEA]o, but the rate constants k-1 and k1 must be extracted from kinetic data for Cd2+ block.
We first estimated the association rate constant for Cd2+ inhibition of IKs assuming that the onset of Cd2+-induced inhibition also obeyed pseudofirst order association kinetics. Fig 6 A shows a typical recording for Cd2+ block of V319Y/F54C currents and a plot of the averaged peak currents versus time of application of Cd2+. The onset of block was well fit by a single-exponential decay function. On average, the onset time constant was 12.5 ± 2.2 s (n = 4) and, thus, the association rate (kapp) was 0.08 s-1. We next determined k-1 from the wash out of Cd2+ block in Fig 6 B. As in the experiment of Fig 4, we first exposed the cell to Cd2+, blocked the channels, and returned it to Cd2+-free solution. After washing out Cd2+, the currents recovered slowly to the control values with averaged time constant of 20.1 ± 1.1 s (n = 4). Assuming pseudofirst order dissociation kinetics, the dissociation rate constant (k-1) is 0.05 s-1. Thus, the association rate constant (k1 = (kapp - k-1)/L) and the apparent dissociation constant (Kd = k-1/k1) were estimated 15 M-1s-1 and 3.3 x 10-3 M, respectively.
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We next began testing for evidence of overlap of TEA+ and Cd2+ binding sites in the channels by studying the influence of externally applied TEA+ on the kinetics of Cd2+ block of expressed channels. As shown in the bottom panels of Fig 6, we found no significant effect of external TEA+ (2 mM) on the onset of or recovery from Cd2+ block of V319Y/F54C channels (onset = 16.4 ± 2.8 s [P = 0.06 versus control; paired t test]; and offset
= 21.5 ± 0.9 s [P = 0.25 versus control; paired t test]). These data suggest that the sites for TEA+ and Cd2+ block do not overlap. We repeated these experiments testing for the effects of extracellular TEA+ on Cd2+ block of a second KCNE1 mutant (G55C), in which the mutated cysteine may be located deeper within the channel pore and, thus, more susceptible to occlusion by TEA+ block of the channel. However, as was the case for V391Y/F54C channels, we found no effect of TEA+ on the on or off kinetics of Cd2+ block of V319Y/G55C channels (Fig 7).
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Finally, to test the predictions of the effects of TEA+ on the kinetics of Cd2+ block more completely, we investigated the effects of a broad range of TEA+ concentrations on Cd2+ block. We measured the ratio of time constants of Cd2+ block in the presence () and absence (
0) of TEA+, which according to Equation 1 HREF="#FD2">Equation 2Equation 3, is given by the following relationship:
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(4) |
where terms are as defined above. In the limit when fUB = 0, or when all channels are blocked by TEA+, this reduces to /
0 =(L/Kd + 1).
The results of our experiments are summarized in Fig 8. First, we confirmed that, at 10 mM [Cd2+]o, expression of V319Y KCNQ1 and F54C KCNE1 encodes Cd2+-sensitive channels. We have found that a 30-s application of 10 mM Cd2+ suppressed the currents by 70.1 ± 3.9% (n = 5), compared with 26.0 ± 9.6% (n = 3) suppression of V319Y/WT channels, indicating significant Cd2+ block conferred by the F54C mutation. Next, we determined the kinetics of the onset of Cd2+ block in the presence and absence of TEA+ and the experiments are summarized in Fig 8. Fig 8 (A and B) illustrates records and summary data from a typical experiment in which we measured the effects of 2 mM TEA+ on Cd2+ block of the channels. In the same cell, we first applied 10 mM Cd2+ for a fixed period (1.5 min; Fig 8 A), and measured the effects of the cation on currents (asterisks) recorded during depolarizing pulses (Fig 8 B). We washed out Cd2+ (closed circle), applied 2 mM TEA+ which blocked the currents by 80% (fUB = 0.20) (arrowhead), and reapplied 10 mM Cd2+ (asterisks in Fig 8 B, top). On average (five cells), we found the fUB value was 0.18 ± 0.01. We then used this value and calculated the /
o ratio in each cell. At 2 mM TEA+, we determined a mean
/
o ratio of 1.1 ± 0.1, which is much smaller than the ratio of 2.6 predicted by Equation 4 using our measured dissociation constant (3.3 mM). Other
/
o ratios were determined with different concentrations of TEA+, and then summarized in Fig 8 D. For comparison, we repeated these experiments for KCNQ1 (WT)/F54C channels over a much higher range of TEA+ concentrations (Fig 1). We found that neither 50 mM TEA+ (fUB = 0.71) nor 100 mM TEA+ (fUB = 0.3) markedly affects the kinetics of Cd2+ block of expressed channels in contrast to the results of similar experiments in Xenopus oocytes (
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DISCUSSION |
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We have used the substitute cysteine accessibility method (
Consideration of Previous Data
Our results contradict the interpretation of previous experimental previous data, obtained primarily in Xenopus oocytes, which suggested that the same cysteine-substituted site (F54C, KCNE1) is exposed deep into the conducting pore (
Association with KCNE1 Protein Does Not Markedly Alter the Structure of the Outer Pore of KCNQ1 Channel
In the present experiments, because we studied channels expressed in CHO cells in which endogenous expression of KCNQ1 subunits is minimal (
Implications for KCNE1/KCNQ1 Assembly
How is it possible to reconcile our data with previous investigations of the location of KCNE1 relative to the KCNQ1 channel pore? In terms of structure and molecular size, it is unlikely that KCNE1 lines the deep pore in the vicinity of the K+ channel selectivity filter (
Implications for Other Channels
KCNE1 is the first member of a family of proteins referred to as KCNE proteins. Coassembly of KCNE1 with KCNQ1 causes marked changes in channel gating and single-channel properties (
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Footnotes |
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1 Abbreviation used in this paper: CHO, Chinese hamster ovary.
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Acknowledgements |
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We thank Dr. J. Pascual for discussion and helpful suggestions with this work.
This work is supported by U.S. Public Health Service Grant HL 44365-5 (to R.S. Kass) and Uehara Memorial Foundation, Japan (to J. Kurokawa).
Submitted: 2 August 2000
Revised: 20 October 2000
Accepted: 21 October 2000
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