Modulation of potassium channel gating by coexpression of Kv2.1 with regulatory Kv5.1 or Kv6.1 alpha -subunits

J. W. Kramer, M. A. Post, A. M. Brown, and G. E. Kirsch

Department of Physiology and Biophysics, Rammelkamp Research Center, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We have determined the effects of coexpression of Kv2.1 with electrically silent Kv5.1 or Kv6.1 alpha -subunits in Xenopus oocytes on channel gating. Kv2.1/5.1 selectively accelerated the rate of inactivation at intermediate potentials (-30 to 0 mV), without affecting the rate at strong depolarization (0 to +40 mV), and markedly accelerated the rate of cumulative inactivation evoked by high-frequency trains of short pulses. Kv5.1 coexpression also slowed deactivation of Kv2.1. In contrast, Kv6.1 was much less effective in speeding inactivation at intermediate potentials, had a slowing effect on inactivation at strong depolarizations, and had no effect on cumulative inactivation. Kv6.1, however, had profound effects on activation, including a negative shift of the steady-state activation curve and marked slowing of deactivation tail currents. Support for the notion that the Kv5.1's effects stem from coassembly of alpha -subunits into heteromeric channels was obtained from biochemical evidence of protein-protein interaction and single-channel measurements that showed heterogeneity in unitary conductance. Our results show that Kv5.1 and Kv6.1 function as regulatory alpha -subunits that when coassembled with Kv2.1 can modulate gating in a physiologically relevant manner.

rat brain; Xenopus oocytes; delayed rectifier; DRK1; IK8; K13

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

VOLTAGE-GATED POTASSIUM channels are transmembrane proteins consisting of four alpha -subunits arranged in radially symmetric fashion around a central aqueous pore. In mammals, a large family of genes encodes alpha -subunits: Kv1 (homologous to Drosophila Shaker), Kv2 (Shab), Kv3 (Shaw), Kv4 (Shal), Kv5, Kv6, and Kv8. Heterologous expression of homotetrameric channels of the Kv1-Kv4 subfamilies display distinctive gating characteristics associated with variations in the primary sequence. In addition, gating is known to be modified through assembly of heterotetramers consisting either of different alpha -subunits (14) or alpha -subunits with accessory beta -subunits (26). Heteromeric assembly has been thought to be restricted to members of the same Kv subfamily (6, 21). However, recent evidence indicates that heteromeric assembly with functional consequences can occur between alpha -subunits from different subfamilies. The Kv2 (Shab) subfamily is particularly noteworthy in this regard. Only two mammalian members have been identified, Kv2.1 and Kv2.2, and both express channels with very similar gating (10, 13). However, when Kv2.1 is coexpressed with certain other alpha -subunits that, by themselves, do not produce electrically functional channels, heteromeric channels with markedly altered gating kinetics are formed (12, 25). Moreover, regulation of gating by electrically silent alpha -subunits is not restricted to the Kv2 subfamily. Recent evidence indicates the existence of regulatory alpha -subunits that interact with members of the Shal (16) and Kv3 (Shaw) (28) subfamilies as well.

We demonstrated previously that coexpression of Kv2.1 with the electrically silent Kv6.1 (formerly known as K13; Ref. 9) results in potassium channels that had slower kinetics of deactivation (25). Evidence for heteromeric assembly was obtained using a yeast two-hybrid assay that demonstrated interactions between the NH2 termini of each alpha -subunit and through a functional assay that showed altered sensitivity to a pore-blocking drug and markedly slower closing rates upon repolarization (deactivation). No change in inactivation kinetics was reported. Recently, however, Kv8.1, Kv6.1 (4, 27, 28), and to a much lesser extent Kv5.1 (Ref. 28; formerly known as IK8; Ref. 9) were shown to slow the time course of Kv2.1 inactivation.

Kv2.1 channels show characteristically slow kinetics of inactivation (5-20 s) that may be related to C-type inactivation (7) originally observed in the Kv1 (Shaker) class of channels. The physiological relevance of this type of inactivation is unclear in view of its extremely slow time course. Its mechanism likewise is uncertain but appears to involve pore-lining residues rather than the cytoplasmic NH2 terminus that is responsible for N-type inactivation (5, 31). Moreover, unlike fast, N-type inactivation in Shaker, slow inactivation in Kv2.1 appears to involve a substantial contribution from a pathway leading directly from closed to inactivated states of the channel (19). Several studies (23, 24) have shown that all four alpha -subunits participate cooperatively in C-type inactivation. Therefore, if each alpha -subunit is involved in the inactivation process, then it is likely that the formation of heteromeric channels can influence the kinetics of inactivation, as was observed in Kv2.1/Kv8.1 coexpression (27). We previously obtained yeast two-hybrid evidence for interactions between Kv2.1 and Kv5.1 but presented no data concerning a functional interaction. Our goal in the present paper was to compare the effects on channel gating of coexpression of Kv5.1 and Kv2.1 with those obtained in Kv2.1/Kv6.1 coexpression. We paid particular attention to regulatory effects of coexpression on the rates of onset and recovery from inactivation over a wide range of voltages. We found that coexpression of Kv5.1 or, to a lesser extent Kv6.1, with Kv2.1 markedly accelerated closed channel inactivation such that both its voltage range and time course became more appropriate for a potential role in regulating membrane excitability.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Yeast two-hybrid system and fusion protein constructs. The MATCHMAKER Yeast Two-Hybrid System (Clontech Laboratories, Palo Alto, CA) was used to assay for protein-protein interactions. The NH2 termini of the voltage-gated potassium channels Kv1.2 nt 1-492 (amino acids 1-164), Kv2.1 nt 1-504 (amino acids 1-169), Kv5.1 nt 1-528 (amino acids 1-176), and Kv6.1 nt 1-627 (amino acids 1-209) were cloned into yeast two-hybrid vectors pGAD424 (LEU2, ampr) and pGBT9 (TRP1, ampr). The Kv channel inserts were generated by PCR using upstream oligonucleotides with an EcoR I site and downstream oligonucleotides with a stop codon/Sal I site to allow directional cloning and correct termination of the fusion protein. All sequences were verified by manual sequencing. Yeast strain Y190 (MATa, Ura3-52, His3-200, Lys2-801, Ade2-101, Trp1-901, Leu2-3, 112, gal4Delta , gal80Delta , cyhr2, LYS2::GAL1UAS-HIS3TATA-HIS3, URA3::GAL1UAS-GAL1TATA-lacZ) (Clontech Laboratories, Palo Alto, CA) was used for all experiments. Y190 cells were transformed with the plasmid constructs of interest (0.1-1 µg of each) and plated on -Trp/-Leu media to select for cells harboring both vectors. LacZ expression assays were performed by lifting Y190 colonies onto grade 410 filter paper (VWR, West Chester, PA), freeze thawing in liquid nitrogen, and incubating at 37°C for up to 6 h on filter paper soaked in Z buffer (in mM: 60 Na2HPO4, 40 NaH2PO4, 10 KCl, and 1 MgSO4, pH 7.0) to which (final concentrations) 0.27% beta -mercaptoethanol and 0.33 mg/ml 5-bromo-4-chloro-3-indoyl-beta -D-galactopyranoside (X-gal) (Research Biochemicals, Natick, MA) had been added. All reagents used, unless otherwise stated, were purchased from Sigma Chemical (St. Louis, MO), Fisher Scientific (Pittsburgh, PA), or Boehringer Mannheim (Indianapolis, IN).

RNA synthesis. Kv2.1, Kv5.1, and Kv6.1 in Bluescript SK- were linearized at the 3' Not I site, proteinase K treated, phenol chloroform extracted, precipitated, and resuspended in water at a concentration of ~1 µg/µl. Linearized cDNA was transcribed in vitro with the mMessage mMachine kit (Ambion, Austin, TX) according to the manufacturer's directions. The final cRNA products were suspended in 100 mM KCl, and their concentration and integrity were checked by formaldehyde agarose gel electrophoresis.

Oocyte preparation. Stage V-VI Xenopus oocytes were defolliculated by collagenase treatment (2 mg/ml for 1.5 h) in a Ca-free buffer solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 5 mM HEPES, and 100 µg/ml gentamicin, pH 7.6). The defolliculated oocytes were injected with 46 nl cRNA solution (in 100 mM KCl) and incubated at 19°C in culture medium (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 2.5 mM pyruvic acid, and 100 µg/µl gentamycin, pH 7.6). Oocytes were injected with 2.5 ng/µl Kv2.1 cRNA for all experiments. In coexpression experiments, 25 ng/µl Kv5.1 or Kv6.1 was coinjected with 2.5 ng/µl Kv2.1.

Whole cell current recording. Standard two-microelectrode voltage-clamp techniques were used to measure macroscopic current (8). Micropipette tip resistance was 0.5-1 MOmega when filled with 3 M KCl plus 1% agar. The bath solution was a normal Ringer solution of the following composition (in mM): 120 NaCl, 2.5 KCl, 2 CaCl2, and 10 HEPES, pH 7.4. In some experiments, 120 mM NaCl was replaced with KCl (K-Ringer solution). Recordings were performed 2-6 days after cRNA injection. Voltage commands and data acquisition were controlled with pCLAMP software (Axon Instruments, Foster City, CA). All measurements were taken at room temperature (21-23°C), and data were filtered at 1 kHz and digitized at 5 kHz. The current traces were corrected for leak off-line except where noted. Data analysis and curve fitting were performed using pCLAMP software. The choice of biexponential over monoexponential functions to fit kinetic data was based on an F ratio test (P < 0.05) that takes into account the increased number of free parameters. Where appropriate, data are expressed as means ± SE. Statistical significance of the difference between means was evaluated using a two-tailed Student's t-test (P < 0.05 or 0.01, as noted).

Single-channel recording. Cell-attached patch recording was performed after manual removal of the vitelline envelope. Isotonic KCl bathing solution was used to zero the resting potential, and the absence of resting membrane potential was verified by rupturing the membrane patch at the end of each experiment to allow direct intracellular potential measurement. Holding and test potentials applied to the membrane patch during the experiment are reported as conventional intracellular potentials. Channels were activated by rectangular test pulses from negative holding potentials. Current records were low pass filtered at 1-2 kHz (-3 dB, 4-pole Bessel filter) then digitized at 5-10 kHz. Linear leakage and capacitative currents were subtracted digitally using the smoothed average of 10-20 null traces in which no channel openings could be detected. Openings were identified using a half-amplitude criterion (Transit analysis program, Ref. 30). Amplitude histograms of the idealized records were fit to Gaussian distributions using a maximum likelihood estimate. Events distributions of <0.3 ms duration were excluded from fitting to avoid error introduced by the limited recording bandwidth (1 kHz).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Coexpression of Kv2.1 with either Kv5.1 or Kv6.1 slows deactivation. We previously showed that coexpression of Kv2.1 and Kv6.1 resulted in currents that deactivated extremely slowly upon repolarization (25). Qualitatively similar results were obtained for coexpression of Kv2.1 and Kv5.1. Figure 1 shows typical records obtained from oocytes that expressed either Kv2.1 (A) or Kv2.1/5.1 (B). Currents were evoked by a two-step protocol consisting of a conditioning step to +60 mV that provided maximum activation, followed by a return to various repolarizing test steps (-100, -80, -60, and -40 mV steps are illustrated). This pulse protocol evoked inward tail currents that subsided with an exponential time course. In both sets of recordings, tail current time course was accelerated by more negative return potentials; however, at test potentials more positive than -80 mV, coexpression of Kv2.1/5.1 (Fig. 1B) resulted in tail currents that decayed more slowly than Kv2.1 (Fig. 1A). Figure 1C shows that the time constant of deactivation for both Kv2.1 (solid circles) and Kv2.1/5.1 (open squares) decreased monotonically with increasingly negative test potentials and that the two curves merged at potentials more negative than -80 mV. At more positive potentials, the curves diverged such that Kv2.1/5.1 time constants were much slower than those of Kv2.1. Our results indicate that heteromer assembly slowed the deactivation process at intermediate but not at extremely negative potentials. This effect was different from that observed previously in Kv2.1/6.1 coexpression (open triangles) in that the magnitude of the Kv2.1/5.1 effect was much smaller and fast deactivation could be restored at sufficiently negative voltage steps.


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Fig. 1.   Effect of Kv5.1 on deactivation. Channels were activated by conditioning pulses to +60 mV that evoked outward current. Repolarizing test pulses of -100 to -20 mV (-100, -80, -60, and -40 are illustrated in A and B) evoked inward tail currents (Im) in oocytes bathed in K-Ringer solution. Linear leakage and capacitative currents were corrected on-line using a P/4 subtraction routine. A: typical records obtained from oocytes injected with Kv2.1 alone. B: records obtained from oocytes coinjected with Kv2.1 and a 10-fold excess of Kv5.1. Decay of tail currents was fit to monoexponential functions, and time constant was plotted as a function of test pulse potential (Em; C). Values are means ± SE (n = 5).

Coexpression potentiates inactivation. During maintained depolarization, channels close by the process of inactivation. Thus, in an oocyte expressing Kv2.1 alone, a test pulse to +40 mV for 15 s (Fig. 2A) evoked a slow, monoexponentially decaying current with a time constant (tau ) of 7 s. Coexpression of Kv2.1 and Kv5.1 alpha -subunits did not alter the onset of inactivation significantly (tau  = 7 s); however, coexpression of Kv2.1 and Kv6.1 resulted in a much slower onset of inactivation (tau  = 32 s) compared with either Kv2.1 alone or Kv2.1/5.1. The onset of inactivation was relatively insensitive to test pulse potential in the range 0 to +40 mV (Fig. 2B) whether Kv2.1 was expressed alone or with either Kv5.1 or Kv6.1. Thus, throughout the voltage range in which channels were strongly activated, the time constants for inactivation were either not significantly affected (Kv2.1/5.1 coexpression) or were markedly increased (Kv2.1/Kv6.1). On this basis, we might be tempted to suggest that coexpression of regulatory subunits with Kv2.1 has, at most, an inhibitory effect on inactivation.


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Fig. 2.   Effects of regulatory subunits on development of inactivation at strongly depolarized test potentials. A: test pulse potentials of +40 mV from a holding potential of -80 mV evoked outward current that decayed exponentially. B: time constants obtained from monoexponential fits of decay of current during long test pulses were plotted on a logarithmic scale vs. test pulse potential for Kv2.1, coexpressed with Kv5.1 or with Kv6.1 (n = 5-8 cells/group).

A very different picture emerged when we investigated inactivation at potentials closer to the foot of the activation range (see Fig. 4B). These experiments used a double-pulse protocol (Fig. 3A, inset) consisting of a depolarizing prepulse of variable duration to initiate inactivation (amplitude, -30 to 0 mV), followed immediately by a +60-mV test pulse that was sufficient to activate all available channels. As shown in Fig. 3A, peak test pulse currents decreased with increasing prepulse duration, such that the envelope of the peaks tracked the time course of inactivation. Peak current was plotted against prepulse length, and data were fit to either monoexponential (Kv2.1) or biexponential (Kv2.1/5.1 and Kv2.1/6.1) decay functions (Fig. 3B). At a prepulse potential of -30 mV, inactivation of Kv2.1 (Fig. 3B, solid circles) was quite slow with a tau  of ~24 s. However, the onset of inactivation was accelerated ~100-fold when Kv2.1 was coexpressed with Kv5.1 (Fig. 3B, open squares) and ~10-fold by Kv6.1 (Fig. 3B, open triangles). Over the range of potentials from -30 to 0 mV, the tau  of inactivation of Kv2.1 was characteristically voltage dependent (Fig. 3C, semilogarithmic plot) such that it became progressively shorter with more positive prepulse potentials. In contrast, for both Kv2.1/5.1 and Kv2.1/6.1 coexpression (Fig. 3, C and D), two time constants were required for an accurate fit, and their voltage dependence was reduced. In Kv2.1/5.1, both fast and slow components of inactivation were significantly faster than the single component of Kv2.1. However, in Kv2.1/6.1, although the fast component was significantly faster than Kv2.1 (Fig. 3C), the slow component (Fig. 3D) was either slower (test potentials, -10 and 0 mV), unchanged (test potential -20 mV), or slightly faster (test potential -30 mV) than Kv2.1 alone.


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Fig. 3.   Regulatory subunits accelerate inactivation at low depolarization. A: typical records obtained from an oocyte expressing Kv2.1 alone using stimulus protocol shown in inset; a variable-length, constant-amplitude (-30 mV is illustrated) conditioning pulse was immediately followed by a short +60-mV test pulse to assess amount of inactivation that developed during conditioning prepulse. Holding potential was -80 mV. B: peak test pulse current vs. prepulse duration for Kv2.1, Kv2.1/6.1, and Kv2.1/5.1 at a prepulse amplitude of -30 mV. Smooth curves are monoexponential (Kv2.1) or biexponential (Kv2.1/5.1 and Kv2.1/6.1) fits to data. C: fast time constant component vs. prepulse amplitude Kv2.1, Kv2.1/5.1, and Kv2.1/6.1 at prepulse potentials of 0, -10, -20, and -30 mV plotted semilogarithmically. Values are means ± SE (n = 4 or 5). All time constants for Kv2.1/5.1 and Kv2.1/6.1 are significantly different from Kv2.1 (P < 0.01). D: slow time constant component vs. prepulse amplitude. Because of its monophasic decay, Kv2.1 data are same in both C and D. Compared with Kv2.1, time constants obtained in Kv5.1 and Kv6.1 coexpression experiments were either significantly different at P < 0.01 (**) or P < 0.05 (*) levels or not significantly different (ns). Each component in Kv5.1 and Kv6.1 data had a relative amplitude of at least 0.2.

The kinetic results obtained at the two different voltage ranges suggested that transitions into the inactivated state from weakly activated, closed states were markedly potentiated in Kv5.1/2.1 and, to a lesser degree, in Kv6.1/2.1 coexpression. Moreover, the voltage dependence of inactivation appeared to be altered by coexpression. To test these ideas further, steady-state inactivation curves were obtained using a two-pulse protocol consisting of a fixed-duration (40 s) prepulse that ranged from -120 to +20 mV in amplitude. After the prepulse, a +40-mV test potential was applied to assess the fraction of noninactivated channels. Figure 4A plots normalized test pulse current vs. prepulse voltage. The data were fit to Boltzmann functions (smooth curves). Coexpression of Kv2.1 with either Kv5.1 or Kv6.1 caused a negative shift in the voltage dependence of the steady-state inactivation curves as compared with Kv2.1. Inactivation in Kv2.1/5.1 showed a half-maximal potential (V0.5) of -57 mV, while Kv2.1/6.1 had a V0.5 of -66 mV. These values differed considerably from Kv2.1, which had a V0.5 of -30 mV. The inactivation curves also differed in steepness of slope. Coexpression of Kv2.1/5.1 and, to a lesser extent, Kv2.1/6.1 tended to reduce the steepness of the curves. (Slope factors of 4.8, 6.4, and 11.1 mV, respectively, were required to fit data from Kv2.1, Kv2.1/6.1, and Kv2.1/5.1.)


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Fig. 4.   Regulatory subunits cause a negative shift of voltage range for inactivation. A: steady-state inactivation curves obtained with conditioning potentials of -120 mV to +10 mV for 40 s and test potential to +40 mV. Peak currents (normalized to maximum observed current in each cell) for Kv2.1, Kv2.1/5.1, and Kv2.1/6.1 are plotted vs. conditioning membrane potential. Values are means ± SE (n = 4 or 5). Smooth curves are fits to a Boltzmann function: h0 + (1 - h0)/{1 + exp[(V - V0.5)/k]}, where h0 is nonzero foot, V is conditioning potential, V0.5 is midpoint potential, and k is a slope factor. Fitted parameters for Kv2.1 were V0.5 = -30.1 mV and k = -4.8 mV. Values for Kv2.1/5.1 were V0.5 = -56.6 mV and k = -11.1 mV. Values for Kv2.1/6.1 were V0.5 = -65.9 mV and k = -6.4. B: steady-state activation curves were obtained from current-voltage families using a series of 500-ms test pulses from -70 to +80 mV from a holding potential of -80 mV. Normalized conductance was estimated from isochronal (2 ms) tail currents at -80 mV. These experiments were performed in K-Ringer solution. Values are means ± SE (n = 4-6). Smooth curve represents Boltzmann fits to data as discussed in text. Fitted parameters for Kv2.1 were V0.5 = -1.7 mV and k = 9.6 mV. Values for Kv2.1/5.1 were V0.5 = 18.5 mV and k = 17.3 mV. Values for Kv2.1/6.1 were V0.5 = -9.4 mV and k = 11.8 mV.

Shifts in the steady-state inactivation curves along the voltage axis (Fig. 4A) could not be predicted from the shifts of the activation curves because as shown in Fig. 4B, the voltage range for Kv2.1/5.1 activation was shifted in a direction opposite to that of the inactivation curve (Fig. 4A). In Kv2.1/6.1, both curves were shifted in the same direction, but inactivation (-35 mV shift) was affected to a much greater degree than activation (-8 mV shift). These data indicate that a major effect of coexpression was to shift the range of potentials in which inactivation occurred to a much more negative region relative to that of activation, consistent with the hypothesis that coexpression promotes inactivation from partially activated closed states of the heteromeric channels.

Coexpression speeds recovery from inactivation. Because coexpression of regulatory subunits had a marked effect on the development of inactivation, we determined whether recovery from inactivation also was affected. Recovery was measured using a four-pulse protocol (Fig. 5A) that consisted of a brief initial prepulse (P1) to assess the maximum available current, followed by a conditioning pulse to 0 mV for 10-15 s to elicit a substantial amount of inactivation. Inactivated channels were allowed to recover for a varying length of time at voltages ranging from -120 to -60 mV before application of a test pulse (P3) to +40 mV. The fractional recovery from inactivation was determined as IP3/IP1. Figure 5B plots the time course of recovery at -90 mV. Points were fit with a monoexponential function to obtain time constants of recovery. Coexpression of Kv2.1 with either Kv5.1 or Kv6.1 accelerated the time course of recovery from inactivation at -90 mV. Average recovery tau  values were 0.8 and 0.3 s for Kv2.1/6.1 and Kv2.1/5.1, respectively, compared with 1.6 s for Kv2.1. Figure 5C shows tau  values of recovery over a wide range of potentials. Recovery from inactivation for Kv2.1/5.1 was consistently faster than Kv2.1 throughout the range tested (-120 to -60 mV, tau  = 0.2-0.8 s). Over the range of potentials tested, Kv2.1 and Kv2.1/6.1 showed a 20-fold change in the time constant as compared with a 4-fold change for Kv2.1/5.1.


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Fig. 5.   Effect of regulatory subunits on recovery from inactivation. A: a test pulse to +40 mV (P1, inset) for 0.25 s (to determine maximum current) was followed by a conditioning pulse (P2) to 0 mV (Kv2.1 and Kv2.1/5.1) or -10 mV (Kv2.1/6.1). After a variable duration (10 ms to 25 s) recovery interval at a constant amplitude of -120 to -60 mV, another test pulse (P3) to +40 mV assessed amount of recovery. B: P2 duration at -90 mV vs. amount of recovery (IP3/IP1). Data were fit with a single exponential equation to obtain time constants of recovery. C: time constants vs. P2 amplitude, plotted semilogarithmically. Values are means ± SE (n = 4-8).

Kv5.1 facilitates cumulative inactivation. Potassium channels often show cumulative inactivation (1), defined as a progressive decline in the amplitude of test pulse currents that occurs during a train of repetitive depolarizing test pulses, each of which is too brief to allow observable inactivation during the pulse. Therefore, we compared the time course of cumulative inactivation in Kv2.1, with and without expression of regulatory subunits, under conditions that would be appropriate for repetitive firing in excitable cells. Figure 6A shows that for Kv2.1/5.1 coexpression, a rapid decline in the amplitude of successive test pulse currents was observed during a stimulus train consisting of short (40 ms) depolarizations to +40 mV, each pulse separated by a 20-ms period at the holding potential of -80 mV (Fig. 6A, inset). Under the same conditions, Kv2.1 and Kv2.1/6.1 showed much slower cumulative inactivation (Fig. 6B). Plots of normalized test pulse current vs. time were accurately fit to monoexponential decay functions with tau  values of 1.6 and 1.2 s, respectively, for Kv2.1 and Kv2.1/6.1. Cumulative inactivation of Kv2.1/5.1, in contrast, followed a biexponential decay with fast and slow tau  values, respectively, of 0.1 and 1.7 s. Continuous depolarization at +40 mV, by comparison, yielded inactivation tau  values of 7 s in Kv2.1 and Kv2.1/5.1 and 32 s in Kv2.1/6.1 (Fig. 2A). Therefore, in all cases, inactivation that proceeds from preopen closed states, which are populated during the rising phase of the test pulse current (note that a 40-ms test pulse achieved <85% of full activation), was more rapid than that which proceeds from open channels. Moreover, Kv2.1/5.1 coexpression was particularly effective in promoting this form of inactivation.


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Fig. 6.   Coexpression of Kv2.1/5.1 accelerates cumulative inactivation. A: typical current traces obtained from Kv2.1/5.1 by repetitive pulsing (inset). Currents evoked by first 10 pulses in train are superimposed; successive test pulses produced progressively smaller amplitude currents until a steady state was reached after pulse 4. For clarity, capacitative transient at beginning of recording (time = 0-1.8 ms) has been blanked. B: peak current vs. elapsed time from start of pulse train. Currents were normalized to initial test pulse current amplitude. Data points are means ± SE (n = 5). Smooth curves show monoexponential [Kv2.1 and Kv2.1/6.1, time constant (tau ) = 2 s] or biexponential fits (Kv2.1/5.1, tau fast = 0.1 s, relative amplitude 0.5; and tau slow = 1.7 s, relative amplitude 0.2).

Kv5.1 and Kv6.1 NH2 termini interact specifically with Kv2.1 NH2 terminus. Yeast two-hybrid assays allow detection of protein-protein interactions by bringing a transcription factor's DNA binding and activation domains together, allowing the subsequent transcription of a reporter gene. The two parts of the transcription factor are brought together by creating fusion proteins with, in this case, portions of Kv channels that may interact. To test for these interactions, X-gal assays were performed on Y190 cells harboring combinations of the activation domain and DNA binding domain in fusion with Kv2.1, Kv5.1, and Kv6.1 as well as the parental pGAD424 and pGBT9 plasmids (Table 1). Interaction strength was determined qualitatively from the intensity and speed of color development. Regardless of the vector background (i.e., DNA binding domain or activation domain), Kv2.1 strongly interacted with itself, Kv5.1, and Kv6.1 (indicated by ++). None of the Kv channel constructs alone was positive (no color development, indicated by -), nor did Kv6.1 interact with itself. In addition, Kv1.2 failed to interact with any of the Kv channels tested except itself. The NH2 termini of Kv5.1, however, did show a slight self-interaction (indicated by -/+). These results confirm our previous report (25) and extend our observations to include interactions between Kv2.1 and Kv5.1.

                              
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Table 1.   X-gal assay of Y190 yeast cells harboring activation domain and DNA binding domain fusion protein constructs of K channel NH2 termini

Heteromeric assembly of Kv2.1/5.1 channels. We previously demonstrated the participation of Kv6.1 in the assembly of Kv2.1/6.1 heteromers based on evidence that coinjection of the two alpha -subunits results in channels with markedly altered pore properties (25). We showed that Kv2.1/6.1 was resistant to block by tetraethylammonium (TEA), presumably because Kv6.1 contains a valine residue in place of the tyrosine, present in both Kv2.1 and Kv5.1, that is required for high sensitivity to block by TEA (11). As predicted by the presence of the critical tyrosine in Kv5.1, no difference in TEA block was observed between Kv2.1 and Kv2.1/Kv5.1 (data not shown). However, single-channel conductance also is regulated by the interaction of all four subunits that make up the pore in heterotetrameric channels (17). Therefore, we measured single-channel conductance as an assay for changes in pore properties that might be attributable to heteromeric Kv2.1/Kv5.1 channel formation. Figure 7, A-C, shows typical single-channel records obtained from cell-attached patches exposed externally to 120 mM KCl solution to maximize the conductance (18). Under these conditions (Fig. 7D), Kv2.1 channels had a conductance of 14.8 pS, whereas in Kv2.1/5.1-injected oocytes, we observed two classes of channels: 12.4 pS (all 13 patches) and 4.2 pS (6 of 13 patches). The differences between the 14.8- and 12.4-pS as well as the 12.4- and 4.2-pS conductance levels were statistically significant (P < 0.04). Therefore, we interpret the 12.4-pS channels as a heteromer of Kv2.1 + Kv5.1, and the 4.2-pS conductance suggests the possibility of multiple classes of heteromeric channels arising from different mixtures of subunits. It is noteworthy that the multiple time constants obtained from analysis of macroscopic inactivation experiments (Fig. 3, C and D) also support the idea of channel heterogeneity.


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Fig. 7.   Effect of Kv5.1 on single-channel conductance. A-C: typical records obtained in cell-attached patches exposed externally to 120 mM KCl solution, in oocytes expressing Kv2.1 alone (A) or Kv2.1 + Kv5.1 (B and C). Data are pooled from 7 Kv2.1 patches and 13 Kv2.1 + Kv5.1 patches that were injected with an RNA ratio of 1 Kv2.1:10 Kv5.1 to increase probability of observing heterotetramers. Test pulses to +80 mV evoked single-channel openings of 1.2 pA in Kv2.1 (A) but only 1.0 pA in Kv2.1/5.1 (B). We observed 2 classes of channels in all patches from Kv2.1 + Kv5.1-injected oocytes: 12.4 pS (all 13 patches) and 4.2 pS (6 of 13 patches). Presence of multiple classes in coexpression is consistent with different combinations of subunits during channel assembly. Kv2.1 channels averaged 14.8 pS (n = 7). Records were obtained from a holding potential of -80 mV. Data were low-pass filtered at 1 kHz. D: single-channel current-voltage plots from 3 classes of channels. Values are means ± SE (n = 7, 13, and 6, respectively, for 15-, 12-, and 5-pS channels). Data are fit by linear regression to obtain single-channel conductances. Difference between 14.8- and 12.4-pS conductance levels was statistically significant (P < 0.05).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Modulation of gating by regulatory alpha -subunits. We have presented here the first evidence of heteromeric assembly of Kv2.1/5.1 channels and the first detailed kinetic observations of the effects of heteromultimerization of Kv2.1 alpha -subunits with electrically silent Kv5.1 and Kv6.1 alpha -subunits. The two subunits can be distinguished on the basis of how they affect the rate of channel closure via two gating reactions: deactivation upon repolarization and inactivation during maintained depolarization (Table 2). Our results show clearly that Kv5.1 accelerates inactivation but has relatively little effect on deactivation. Kv6.1 accelerates inactivation less effectively but has a pronounced slowing effect on deactivation. A separate question is whether the effects on inactivation are associated with pathways involving either closed or open states of the channels. Klemic et al. (19) proposed that in Kv2.1 channels transitions from preopen closed states to the inactivated state are much faster than those from open to the inactivated states. Our observations support this idea by showing that coexpression of Kv2.1 with Kv5.1 markedly accelerated inactivation at intermediate potentials near the foot of the activation curve (-30 to -10 mV) but had almost no effect on the rate of inactivation at strong depolarizations approaching the plateau region (>40 mV). Moreover, coexpression of Kv2.1 with Kv6.1 caused a marked slowing of inactivation at strong depolarizations (Fig. 2A) but acceleration at intermediate potentials (Fig. 3B). These results indicate that, for Kv2.1, two pathways for inactivation exist and that they can be regulated independently of one another.

                              
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Table 2.   Functional characteristics of regulatory alpha -subunits coexpressed with Kv2.1

Two lines of evidence support the notion that these kinetic effects are the result of coassembly of Kv2.1 and Kv5.1 or 6.1 subunits into a functional channel rather than a regulatory effect of Kv5.1 or Kv6.1 on the gating properties of homotetrameric Kv2.1 channels, such as that of beta -subunits on voltage-gated potassium or sodium channels, (26, 15). First, the yeast two-hybrid assay provided evidence for protein-protein interactions between the NH2 termini of these subunits. It is noteworthy that these interactions showed a high degree of specificity; only interactions between Kv2.1 and Kv5.1 or Kv6.1 were observed. Second, single-channel recordings in oocytes coinjected with Kv2.1 and Kv5.1 provided evidence for channels with unitary conductance distinct from that observed in oocytes injected with Kv2.1 alone. This observation indicates a change in pore structure that can most readily be explained by heterotetramer formation.

Electrophysiological analysis of Kv2.1/5.1 coexpression revealed currents with unique inactivation kinetics; the onset of inactivation at strongly depolarized potentials was unchanged, whereas a marked acceleration was observed at potentials near the foot of the activation curve. These results indicated that inactivation from partially activated channels was potentiated. This was further substantiated by a marked shift of the steady-state inactivation curve in the hyperpolarized direction. A potentially important consequence of the regulatory influence of Kv5.1 was revealed in a comparison of the time course of cumulative inactivation during repetitive stimulation, which showed that Kv2.1/5.1 inactivated much more rapidly than either Kv2.1/6.1 or Kv2.1 alone. However, the kinetics of recovery from inactivation also were much faster in Kv2.1/5.1 than in Kv2.1. Because both entry into and recovery from inactivation were accelerated, the balance between these two opposing tendencies would be expected to govern the relationship between the rate of cumulative inactivation and stimulus frequency. At low stimulus frequencies where Kv2.1/5.1 channels have a greater chance to recover from inactivation, the rate of cumulative inactivation would be expected to be slower as compared with high stimulus frequency, where inactivation would be expected to dominate over recovery. Thus the kinetic effect of Kv5.1 on Kv2.1/5.1 heteromers appeared to be directed primarily toward acceleration of the on and off rates of inactivation and a negative shift of its steady-state voltage dependence. This result is in marked contrast to a recently published study (28) that concluded, based on results obtained only with strong depolarizing pulses, that the primary regulatory influence of Kv5.1 (and Kv6.1) is to slow the kinetics of inactivation. In addition, Kv2.1/5.1 heteromers also displayed altered activation kinetics that would tend to hold channels open at physiologically relevant membrane potentials (-80 to -20 mV). Taken together, the modulatory effects associated with Kv2.1/5.1 heteromers would allow greater flexibility in the gating of delayed rectifier channels over a voltage range that is critical for control of membrane excitability.

In contrast, inactivation may be secondary to activation gating as the regulatory target for Kv6.1 in coexpression with Kv2.1. We base this on our previous finding (25) that deactivation was slowed by a factor of 5- to 15-fold over the range -60 to -100 mV and the steady-state voltage dependence of activation was shifted toward more negative potentials. Both of these effects would tend to increase potassium conductance over a physiologically significant potential range corresponding to the voltage region encompassing the action potential threshold and the peak of the hyperpolarizing afterpotential. The effects on inactivation, although qualitatively similar to those of Kv5.1, were much smaller in magnitude and, as a result, had little effect on cumulative inactivation.

Recent studies indicate that, like Kv5.1 and 6.1, the electrically silent Kv8.1 subunit also functions as a regulatory alpha -subunit of Kv2.1 (12). Although initial reports stressed an inhibitory effect of Kv8.1 on Kv2.1 expression (12), more recent studies (4, 27) suggest modulation of channel gating as its primary function. The effects of Kv8.1 on Kv2.1 (or Kv2.2) gating most closely resemble those of Kv5.1 (Table 2). Thus Kv8.1 shifts the voltage dependence of inactivation by approximately -40 mV, whereas activation is shifted by less than +10 mV (4, 27). Like Kv5.1, Kv8.1 produces a relatively small slowing of deactivation (4). However, unlike Kv5.1, Kv8.1 produces a marked slowing of inactivation during strong depolarizing test pulses (27). In this regard, the Kv8.1 kinetic effect more closely resembles that of Kv6.1. It should be noted, however, that Kv6.1's inhibitory effect on inactivation at strongly depolarized potentials was reversed at intermediate test potentials. Whether Kv8.1 has similar characteristics has not been determined.

Implications for inactivation gating. The mechanism of slow inactivation in potassium channels is not thoroughly understood. Our results, however, place important constraints on gating models. Inactivation of delayed rectifier potassium current in molluscan neurons has been described as a voltage- and state-dependent process where the transition to the inactivated state from closed states is more favorable than from the open state (1). Similarly, for Kv2.1 expressed in oocytes, slow inactivation has been modeled as a state-dependent process that is most accessible from a nonconducting, preopen state (19). Our results indicate that, in Kv2.1, the inactivation process for fully activated open channels is regulated differently from that which occurs in weakly activated closed channels. For instance, Kv2.1/6.1 coexpression results in channels with markedly slower inactivation during strong depolarization but faster inactivation at intermediate potentials. Similarly, in Kv2.1/5.1 coexpression, the time course of inactivation during strong depolarization was not affected, whereas inactivation at potentials near activation threshold was accelerated 100-fold. These results are consistent with the notion that inactivation is highly state dependent such that inactivation from channels that are predominantly in the open state can be much slower than inactivation from channels in preopen closed states (19). Thus, regardless of the effect of Kv5.1 or Kv6.1 subunits on the time constant of inactivation during long-lasting, strong depolarizing pulses, the effect on inactivation kinetics at negative test potentials was always one of facilitation, as evidenced by a negative shift in the steady-state voltage dependence of inactivation and faster time constants for both onset and recovery. A second implication concerning mechanisms of inactivation relates to the idea of coupling between the inactivation and activation processes. In voltage-gated sodium channels, the two processes are coupled such that inactivation derives its voltage dependence through a tight coupling to activation (2). Tight coupling also can be detected in delayed recovery imposed by the necessity for channels to deactivate before recovery can proceed upon repolarization (20). In contrast, our results suggest a much looser coupling in Kv2.1. Thus coexpression of Kv2.1/5.1 gave a +15-mV shift in the voltage dependence of activation that was accompanied by a -30-mV shift of the inactivation curve. Similarly, for Kv2.1/6.1, the deactivation process was slowed by a factor of 10, whereas the recovery from inactivation showed either no change or a modest acceleration. These results together with the observation of a "foot" or "U-shaped" configuration of the steady-state inactivation curve (19) suggest that in Kv2.1 recovery from inactivation can take place from open channels and that closed channels can inactivate.

Potential physiological significance of alpha -subunit regulation. Functional diversity achieved through assembly of heteromeric channels from phenotypically distinct alpha -subunits is a hallmark of the Kv1 and Kv3 subfamilies. In contrast, the Kv2 subfamily has only two phenotypically indistinguishable mammalian members (Kv2.1 and Kv2.2). Our results suggest that in this subfamily functional diversity is promoted through heteromeric channel assembly with Kv5.1 and Kv6.1 alpha -subunits. Moreover, the regulatory influence of these subunits is clearly distinguishable: Kv5.1 primarily accelerates inactivation, whereas Kv6.1 primarily slows deactivation. The potential physiological consequences can be inferred from measurement of cumulative inactivation during repetitive pulsing. Under conditions that simulate high-frequency trains of brief impulses, we found that Kv2.1/5.1 but not Kv2.1/6.1 showed a marked acceleration of the rate of cumulative inactivation. Slow recovery from inactivation has also been suggested as a factor in promoting cumulative inactivation (3). However, under our stimulus regimen (40-ms depolarizing pulses, 20-ms interpulse intervals), changes in recovery rates cannot play a role because no significant recovery occurred during the brief interpulse interval. Under these experimental conditions, the rapidity of inactivation during the rising phase of the test pulse currents, rather than differences in recovery during the interpulse interval, was the major determinant. Thus the much greater acceleration of inactivation in Kv2.1/5.1 than in Kv2.1/6.1 may account for the effect on cumulative inactivation. Cumulative inactivation in neurons is thought to be an important determinant patterning repetitive spike discharges (29) and in frequency-dependent spike broadening duration of trains of repetitive pulses (22). Our results suggest that a switch from either predominantly Kv2.1 homotetramers or Kv2.1/6.1 heterotetramers to more rapidly inactivating Kv2.1/5.1 heterotetramers would increase the excitability of neurons during repetitive firing by increasing spike frequency and prolonging the action potential. On the other hand, compared with Kv2.1 homotetramers, Kv2.1/6.1 heterotetramers require a much longer time to close upon repolarization and, therefore, might be expected to prolong the refractory period that determines the interspike interval and thereby reduce spike frequency during repetitive firing.

    ACKNOWLEDGEMENTS

We thank W.-Q. Dong and C.-D. Zuo for expert oocyte injection and culture.

    FOOTNOTES

This work was supported by National Institutes of Health Grants NS-29473 (to G. E. Kirsch), NS-23877, HL-36930, and HL-55404 (to A. M. Brown); National Research Service Award 93849 (to M. A. Post); and a grant-in-aid from the American Heart Association, Northeast Ohio Affiliate (to J. W. Kramer).

Address for reprint requests: G. E. Kirsch, MetroHealth Medical Center, Rammelkamp Bldg. R327, 2500 MetroHealth Dr., Cleveland, OH 44109.

Received 22 October 1997; accepted in final form 19 February 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Aldrich, R. W., P. A. Getting, and S. H. Thompson. Mechanism of frequency-dependent broadening of molluscan neurone soma spikes. J. Physiol. (Lond.) 291: 531-544, 1979[Abstract].

2.   Armstrong, C. M., and F. Bezanilla. Inactivation of the sodium channel. II. Gating current experiments. J. Gen. Physiol. 70: 567-590, 1977[Abstract].

3.   Bertoli, A., O. Moran, and F. Conti. Accumulation of long-lasting inactivation in rat brain K+-channels. Exp. Brain Res. 110: 401-412, 1996[Medline].

4.   Castellano, A., M. D. Chiara, B. Mellstrom, A. Molina, F. Monje, J. R. Naranjo, and J. Lopez-Barneo. Identification and functional characterization of a K+ channel alpha -subunit with regulatory properties specific to brain. J. Neurosci. 17: 4652-4661, 1997[Abstract/Free Full Text].

5.   Choi, K. L., R. W. Aldrich, and G. Yellen. Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. Proc. Natl. Acad. Sci. USA 88: 5092-5095, 1991[Abstract].

6.   Covarrubias, M., A. A. Wei, and L. Salkoff. Shaker, Shal, Shab, and Shaw express independent K+ current systems. Neuron 7: 763-773, 1991[Medline].

7.   De Biasi, M., H. A. Hartmann, J. A. Drewe, M. Taglialatela, A. M. Brown, and G. E. Kirsch. Inactivation determined by a single site in K+ pores. Pflügers Arch. 422: 354-363, 1993[Medline].

8.   Drewe, J. A., H. A. Hartmann, and G. E. Kirsch. K+ channels in mammalian brain: a molecular approach. Methods Neurosci. 19: 243-260, 1994.

9.   Drewe, J. A., S. Verma, G. Frech, and R. H. Joho. Distinct spatial and temporal expression patterns of K+ channel mRNAs from different subfamilies. J. Neurosci. 12: 538-548, 1992[Abstract].

10.   Frech, G. C., A. M. J. VanDongen, G. Schuster, A. M. Brown, and R. H. Joho. A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning. Nature 340: 642-645, 1989[Medline].

11.   Heginbotham, L., and R. Mackinnon. The aromatic binding site for tetraethylammonium ion on potassium channels. Neuron 8: 483-491, 1992[Medline].

12.   Hugnot, J. P., M. Salinas, F. Lesage, E. Guillemare, J. de Weille, C. Heurteaux, M. G. Mattei, and M. Lazdunski. Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties toward Shab and Shaw channels. EMBO J. 15: 3322-3331, 1996[Abstract].

13.   Hwang, P. M., C. E. Glatt, D. S. Bredt, G. Yellen, and S. H. Snyder. A novel K+ channel with unique localizations in mammalian brain: molecular cloning and characterization. Neuron 8: 473-481, 1992[Medline].

14.   Isacoff, E. Y., Y. N. Jan, and L. Y. Jan. Evidence for the formation of heteromultimeric potassium channels in Xenopus oocytes. Nature 345: 530-534, 1990[Medline].

15.   Isom, L. L., K. S. De Jongh, and W. A. Catterall. Auxiliary subunits of voltage-gated ion channels. Neuron 12: 1183-1194, 1994[Medline].

16.   Jegla, T., and L. Salkoff. A novel subunit for Shal K+ channels radically alters activation and inactivation. J. Neurosci. 17: 32-44, 1997[Abstract/Free Full Text].

17.   Kirsch, G. E., J. A. Drewe, M. De Biasi, H. A. Hartmann, and A. M. Brown. Functional interactions between K+ pore residues located in different subunits. J. Biol. Chem. 268: 13799-13804, 1993[Abstract/Free Full Text].

18.   Kirsch, G. E., J. M. Pascual, and C. C. Shieh. Functional role of a conserved aspartate in the external mouth of voltage-gated potassium channels. Biophys. J. 68: 1804-1823, 1995[Abstract].

19.  Klemic, K. G., C. C. Shieh, G. E. Kirsch, and S. W. Jones. Inactivation of Kv2.1 potassium channels. Biophys. J. In press.

20.   Kuo, C. C., and B. P. Bean. Na+ channels must deactivate to recover from inactivation. Neuron 12: 819-829, 1994[Medline].

21.   Li, M., Y. N. Jan, and L. Y. Jan. Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channel. Science 257: 1225-1230, 1992[Medline].

22.   Ma, M., and J. Koester. The role of K+ currents in frequency-dependent spike broadening in Aplysia R20 neurons: a dynamic-clamp analysis. J. Neurosci. 16: 4089-4101, 1996[Abstract/Free Full Text].

23.   Ogielska, E. M., W. N. Zagotta, T. Hoshi, S. H. Heinemann, J. Haab, and R. W. Aldrich. Cooperative subunit interactions in C-type inactivation of K channels. Biophys. J. 69: 2449-2457, 1995[Abstract].

24.   Panyi, G., Z. Sheng, and C. Deutsch. C-type inactivation of a voltage-gated K+ channel occurs by a cooperative mechanism. Biophys. J. 69: 896-903, 1995[Abstract].

25.   Post, M. A., G. E. Kirsch, and A. M. Brown. Kv2.1 and electrically silent Kv6.1 potassium channel subunits combine and express a novel current. FEBS Lett. 399: 177-182, 1996[Medline].

26.   Rettig, J., S. H. Heinemann, F. Wunder, C. Lorra, D. N. Parcej, J. O. Pongs, and O. Pongs. Inactivation properties of voltage gated K+ channels altered by presence of beta -subunit. Nature 369: 289-294, 1994[Medline].

27.   Salinas, M., J. de Weille, E. Guillemare, M. Lazdunski, and J. P. Hugnot. Modes of regulation of shab K+ channel activity by the Kv8.1 subunit. J. Biol. Chem. 272: 8774-8780, 1997[Abstract/Free Full Text].

28.   Salinas, M., F. Duprat, C. Heurteaux, J. Hugnot, and M. Lazdunski. New modulatory alpha  subunits for mammalian Shab K+ channels. J. Biol. Chem. 272: 24371-24379, 1997[Abstract/Free Full Text].

29.   Storm, J. F. Temporal integration by a slowly inactivating K+ current in hippocampal neurons. Nature 336: 379-381, 1988[Medline].

30.   VanDongen, A. M. A new algorithm for idealizing single ion channel data containing multiple unknown conductance levels. Biophys. J. 70: 1303-1315, 1996[Abstract].

31.   VanDongen, A. M., G. C. Frech, J. A. Drewe, R. H. Joho, and A. M. Brown. Alteration and restoration of K+ channel function by deletions at the N- and C-termini. Neuron 5: 433-443, 1990[Medline].


Am J Physiol Cell Physiol 274(6):C1501-C1510
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