Distinct domains of the voltage-gated K+ channel Kvbeta 1.3 beta -subunit affect voltage-dependent gating

Victor N. Uebele1, Sarah K. England2, Daniel J. Gallagher2, Dirk J. Snyders1,3, Paul B. Bennett1,3, and Michael M. Tamkun1,2

Departments of 1 Pharmacology, 2 Molecular Physiology and Biophysics, and 3 Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The Kvbeta 1.3 subunit confers a voltage-dependent, partial inactivation (time constant = 5.76 ± 0.14 ms at +50 mV), an enhanced slow inactivation, a hyperpolarizing shift in the activation midpoint, and an increase in the deactivation time constant of the Kv1.5 delayed rectifier. Removal of the first 10 amino acids from Kvbeta 1.3 eliminated the effects on fast and slow inactivation but not the voltage shift in activation. Addition of the first 87 amino acids of Kvbeta 1.3 to the amino terminus of Kv1.5 reconstituted fast and slow inactivation without altering the midpoint of activation. Although an internal pore mutation that alters quinidine block (V512A) did not affect Kvbeta 1.3-mediated inactivation, a mutation of the external mouth of the pore (R485Y) increased the extent of fast inactivation while preventing the enhancement of slow inactivation. These data suggest that 1) Kvbeta 1.3-mediated effects involve at least two distinct domains of this beta -subunit, 2) inactivation involves open channel block that is allosterically linked to the external pore, and 3) the Kvbeta 1.3-induced shift in the activation midpoint is functionally distinct from inactivation.

Shaker-like potassium channel; N-type inactivation; C-type inactivation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

VOLTAGE-GATED K+ channels represent a structurally and functionally diverse group of membrane proteins. These channels establish the resting membrane potential and modulate the frequency and duration of action potentials in nerve and muscle (18). In addition, K+ channels are involved in processes not usually associated with electrically excitable membranes, such as T lymphocyte activation, cell volume regulation, and pancreatic beta -cell function (5, 22). Multiple Shaker-like K+ channel alpha -subunit genes have been cloned from mammalian brain, heart, skeletal muscle, pancreas, and smooth muscle and functionally expressed in heterologous systems (6). The recent discovery of beta -subunits, some of which convert delayed rectifiers into rapidly inactivating channels, has revealed additional mechanisms of voltage-gated K+ channel modulation (9, 12-14, 17, 26, 28, 32, 33). Functional analysis has shown that four of these beta -subunits, Kvbeta 1.1, Kvbeta 1.2, Kvbeta 1.3, and Kvbeta 3.1, confer varying degrees of rapid inactivation on certain members of the Kv1 family of delayed rectifiers (12, 13, 16, 17, 26, 28, 32, 33). In addition, Kvbeta 1.2, Kvbeta 1.3, and Kvbeta 2.1 modify the voltage dependence of Kv1.5 channel opening (8-20 mV hyperpolarizing shift in the midpoint of activation) (12, 13, 40). However, it has been suggested that the apparent shift in the Kv1.2 activation curve following coexpression with Kvbeta 1.2 is completely derived from the beta -subunit-induced N-type or fast inactivation (10). In this respect, it is important to note that Kvbeta 2.1, which does not induce fast inactivation, does shift the activation midpoint and only enhances slow or C-type inactivation of the Kv1.5 delayed rectifier (40). Each Kvbeta family (Kvbeta 1, Kvbeta 2, and Kvbeta 3) appears to derive from a separate gene, and additional variability in the Kvbeta 1 family results from alternative splicing in the amino-terminal region, thus yielding the Kvbeta 1.1, Kvbeta 1.2, and Kvbeta 1.3 subunits (12, 27). The variable amino-terminal domains are responsible for the functional differences (16, 29, 32), whereas the conserved carboxy-terminal domain most likely governs assembly with the alpha -subunit (16, 30, 34, 43, 45).

The rapid inactivation induced by Kvbeta 1.1 when coexpressed with the delayed rectifier, Kv1.1, is thought to occur by the same "ball-and-chain" mechanism reported for the Shaker channel (20, 32). For example, exposure of the cytoplasmic face of Kv1.1 to a peptide corresponding to the 24 amino-terminal amino acids of Kvbeta 1.1 causes inactivation (32). One interpretation of this finding is that the peptide induces inactivation by binding within the open pore. Alternatively, the peptide could bind to a site removed from the pore and allosterically modulate ion permeation. Indeed, Morales and co-workers (29) have presented evidence that Kvbeta 1.2 confers inactivation on a delayed rectifier by enhancing primarily C-type inactivation (29), suggesting that Kvbeta 1.2-induced inactivation occurs by an allosteric mechanism. Thus no a priori assumptions should be made about the inactivation mechanism of other Kvbeta subunits.

The Kvbeta 1.3 subunit converts Kv1.5 from a delayed rectifier with a modest degree of slow inactivation to a channel with both fast and slow components of inactivation. The rapid inactivation has the characteristics of open channel block and is similar to block of Kv1.5 by antiarrhythmic agents such as quinidine. Mutagenesis of alpha - and beta -subunits followed by kinetic analysis of channel gating was used in the present study to examine the mechanism by which Kvbeta 1.3 modulates Kv1.5 function. The data indicate that Kvbeta 1.3-mediated fast inactivation likely occurs in part by an open channel block mechanism that is sensitive to both membrane potential and external K+. Both the beta -subunit-enhanced slow inactivation and the modulation of beta -subunit-induced fast inactivation by an external, but not an internal, pore mutation reveal the importance of allosteric mechanisms in K+ channel alpha -beta interactions. Results presented here clearly indicate that the Kvbeta 1.3 subunit directly alters alpha -subunit activation, for Kvbeta 1.3 mutants that produce no inactivation still induce a hyperpolarizing shift in the voltage dependence of Kv1.5 activation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Enzymes and buffers were from New England Biolabs (Beverly, MA), Boehringer Mannheim (Indianapolis, IN), and Promega (Madison, WI). The Sequenase 2.0 kit was purchased from United States Biochemical (Cleveland, OH). The origins of other materials are specified below.

Mutagenesis. The series of amino-terminal deletions in Kvbeta 1.3 shown in Fig. 1 was created by PCR mutagenesis. The 5' oligonucleotide primer replaced amino acids 10, 37, 68 and 91, respectively, with an Xba I restriction site and a consensus translation initiation site (GGTCTAGAATG. . .). The 3' oligonucleotide annealed downstream from a unique endogenous Kpn I (Delta N10 and Delta N37) or Pst I (Delta N68 and Delta N91) restriction site. The 5' sequence of the previously described Kvbeta 1.3 construct in the modified pSP64T vector (12) was excised with the appropriate restriction enzymes, and the amplified mutant PCR products were ligated in its place. Two additional mutants were made in which the first 19 or 87 amino acids of Kvbeta 1.3 were linked to the amino terminus of Kv1.5. The 5' region of Kvbeta 1.3 was PCR amplified to contain an Sph I site at the 3' end in the same reading frame as an endogenous Sph I site in the 5' untranslated region of Kv1.5. Use of this site introduced seven amino acids (His-Ala-Leu-Cys-Ser-Arg-Ala) that are not part of either wild-type subunit. The tandem constructs were then ligated into a modified pSP64T vector for oocyte expression. These constructs will be referred to as tandems even though only portions of the amino terminus of Kvbeta 1.3 are appended to the full-length Kv1.5 (Fig. 1). All PCR-generated mutants were verified by double-stranded sequencing. The point mutations in the Kv1.5 alpha -subunit have been described previously (44). These mutants were subcloned into the pSP64T vector for oocyte expression. It was necessary to add ~710 base pairs of the 3' untranslated sequence of Kv1.5 to constructs containing the alpha -subunit to achieve expression levels in excess of 1.5 µA.


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Fig. 1.   Kvbeta 1.3 mutations. PCR-generated deletion and tandem mutant constructs are shown. Solid bar, carboxy-terminal 329 amino acids of Kvbeta 1.3; shaded bar, variable amino-terminal domain of 91 amino acids. Nomenclature for deletions uses Delta  to indicate a deletion and N to indicate that deletion is amino-terminal, and number indicates amino acid that was replaced with the initiating methionine. Tandem constructs link coding sequence of Kvbeta 1.3 amino terminus with coding sequence of complete Kv1.5. Nomenclature for tandems uses N to indicate that amino terminus of Kvbeta 1.3 was added to amino terminus of Kv1.5, and number indicates number of Kvbeta 1.3 amino acids added, as indicated by shaded bar. Circle, 7 amino acids (His-Ala-Leu-Cys-Ser-Arg-Ala) inserted between beta - and alpha -subunit domains; shaded boxes, transmembrane domains within alpha -subunit.

Expression in Xenopus oocytes and electrophysiological recording. Templates were linearized with EcoR I before in vitro cRNA synthesis using the SP6 mMessage mMachine kit (Ambion) according to the manufacturer's instructions. Defolliculated Xenopus oocytes were prepared as described previously (12, 13) and injected with ~40 nl (4-20 ng) of in vitro-transcribed cRNA. The cRNA was diluted to yield peak currents of 1-10 µA.

Oocytes were bathed in one of two extracellular solutions, normal K+ or high K+. Normal K+ contained (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.5 with NaOH). High K+ contained (in mM) 2 NaCl, 96 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.5 with KOH). Equilibration of the bath solution was monitored by the stability of the direction and magnitude of the deactivating tail current at -40 mV. Membrane currents were recorded under voltage-clamp conditions using a two-microelectrode voltage amplifier from Warner Instruments (New Haven, CT), as described previously (12, 13). Current-passing and voltage-recording electrodes were filled with 3 M KCl and had resistances of 0.8-2.0 MOmega . Membrane currents recorded from oocytes expressing currents >11 µA were not used in the data analysis. Likewise, data from oocytes expressing high levels of time-dependent background currents were not analyzed. Data acquisition was via Pulse Control v4.5 running on a Macintosh Power PC 8100, and analysis was performed using customized protocols written in IGOR Pro v2.04. All experiments were performed at room temperature.

The holding potential was -80 mV except when the Kv1.5(V512A) subunit was studied. Due to this mutant's -50-mV threshold of activation, these oocytes were held at -100 mV. The cycle time for all pulse protocols was 10 s or slower to allow full recovery from inactivation between pulses. The standard protocol to obtain current-voltage relationships and activation curves consisted of 200-ms pulses that were imposed in 10-mV increments between -60 and +70 mV, followed by a repolarizing step to -40 mV. The voltage dependence of deactivating currents was analyzed by stepping to various voltages after a 50-ms depolarization to +50 mV to maximally activate the channels.

Pulse protocols and data analysis. Activation curves were obtained from the tail current amplitude 6 ms after repolarization to -40 mV. The voltage dependence of channel opening ("activation curve") was fit with the Boltzmann equation I/Imax = {1 + exp[-(Em - Eh)/k]}-1, in which I is current, Imax is maximum current, Em is membrane potential, Eh is the voltage at which 50% of the channels are open, and k is the slope factor. The impact of inactivation on the generation of activation curves is reviewed in DISCUSSION. The time course of deactivating tail currents was fit with a single exponential by a nonlinear least squares algorithm. Goodness of the fit was judged by visual inspection for nonrandom trends in the residuals of the fit. For steady-state current values, raw data points were averaged over a small time window (2-5 ms).

The extent of slow inactivation was determined using a twin-pulse protocol in which both depolarizing pulses were at +70 mV, with the duration of the first pulse varying. An interpulse step to -80 mV for 50 ms permitted full recovery of fast inactivation while minimizing recovery of slow inactivated channels. Comparison of the peak currents from the first and second depolarizing pulses then allowed quantitation of the slow component of inactivation at the given first pulse duration. Inactivated current that recovered within 50 ms at -80 mV was defined as fast inactivated and that not recovered was defined as slow inactivated (see Fig. 4E). The fraction of slow inactivation was calculated as 1 - (P2/P1), where P2 is the peak current of the second pulse and P1 is the peak current of the first pulse.

A two-pulse protocol was used to investigate the voltage dependence of inactivation. The membrane potential of the first pulse was varied in 10-mV increments, and the second pulse was a step to +70 mV. The duration of the first pulse was either 25 ms or 1 s, depending on whether the fast activation or the combination of fast and slow inactivation was being studied. Fast inactivation, with a time constant of 5.7 ms, is >98% complete at 25 ms. Therefore, minimizing the duration of the first pulse to 25 ms allowed completion of the fast component of inactivation while preventing significant accumulation of slow inactivation. The first pulse was extended to 1 s to examine the combined voltage dependence of fast and slow inactivation. The voltage dependence of inactivation was determined using a model (42) in which the product z · delta  describes the sensitivity of the blocking particle to the transmembrane electrical field. In this case, z represents the effective charge of the blocking particle, and delta  represents the fraction of the transmembrane potential at the blocking site (7, 44). For Kvbeta 1.3, z is unknown, and therefore only the product z · delta  is reported. Although this model can be used to characterize the voltage dependence of inactivation, no evidence exists that the voltage dependence is derived from a charged particle sensing the transmembrane field.

Results are expressed as means ± SE. Specific numbers of experiments (n) are presented in the text. All pooled data were collected from the oocytes of at least two different frogs.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study examined the mechanism by which Kvbeta 1.3 alters Kv1.5 function and determined regions within each subunit that affect their functional interactions. In the presence of the Kvbeta 1.3 subunit, the Kv1.5 current phenotype was changed from a delayed rectifier to one that displayed a rapid, but partial, inactivation (time constant = 5.76 ± 0.14 ms at +50 mV, n = 17; Table 1) that was apparent only at membrane potentials more positive than ~0 mV (see Fig. 2). A slower rate of channel deactivation was also observed in the presence of Kvbeta 1.3 relative to Kv1.5 alone (32.6 ± 1.2 ms vs. 13.7 ± 0.5 ms at -40 mV; Table 1). Additionally, the threshold for Kv1.5 channel activation occurred at more negative membrane potentials, with a parallel 13-mV hyperpolarizing shift in the voltage dependence of activation (Table 1).

                              
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Table 1.   Summary of kinetic and steady-state parameters


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Fig. 2.   Fast inactivation localizes to amino terminus of Kvbeta 1.3. Whole cell current was recorded from Xenopus oocytes injected with Kv1.5 (A), Kv1.5 + Kvbeta 1.3 (B), Kv1.5 + Kvbeta 1.3(Delta N10) (C), and tandem(N87) (D). All currents were elicited by 200-ms depolarizations from a holding potential of -80 mV to test potentials between -60 and +70 mV (in 10-mV increments). Test potential was followed by a repolarizing step to -40 mV to record deactivating currents.

Role of the Kvbeta 1.3 amino-terminal sequence in fast inactivation. The various Kvbeta 1 subfamily members confer different functional effects on Kv1.5 (12, 13, 16, 26, 41). The amino termini of these subunits vary considerably, whereas the 329 carboxy-terminal amino acids are identical. Therefore, we constructed several mutations within the Kvbeta 1.3 amino terminus and examined the effect of these alterations on Kvbeta 1.3-induced inactivation. Mutant Kvbeta 1.3 subunits lacking the first 10, 37, 68 and 91 amino acids of the variable amino terminus were constructed. Two tandem mutants were also made in which the first 19 or 87 amino acids of the amino terminus of Kvbeta 1.3 were linked to the amino terminus of Kv1.5 (Fig. 1). Representative outward currents recorded from oocytes expressing Kv1.5, Kv1.5 + Kvbeta 1.3, Kv1.5 + Kvbeta 1.3(Delta N10), and tandem(N87) are shown in Fig. 2. Kv1.5 displayed a delayed rectifier phenotype when expressed alone (Fig. 2A). Coexpression of the Kv1.5 subunit with wild-type Kvbeta 1.3 resulted in currents that exhibit fast inactivation at membrane potentials positive to +10 mV but failed to show any apparent inactivation at more negative potentials (Fig. 2B). At +70 mV, the extent of inactivation at 200 ms, defined as the decline from the peak current level, averaged 54.4 ± 1.1% (n = 16; Table 1). The time constant of Kvbeta 1.3-mediated inactivation at +70 mV was 5.7 ± 0.1 (n = 17; Table 1) and that for recovery from fast inactivation at -80 mV was 5.1 ± 0.1 ms (n = 7). Removal of as few as 10 amino acids from the amino terminus of Kvbeta 1.3 prevented fast inactivation (Fig. 2C). The other more extensive Kvbeta 1.3 amino-terminal deletions also did not induce fast inactivation (data not shown). In contrast to the currents observed with Kv1.2 and amino-terminal-truncated Kvbeta 1.2 (1), no significant increase in outward current was observed following the removal of Kvbeta 1.3-induced inactivation. Therefore, the ratio of the peak to steady-state current shown in Fig. 2B is likely to represent a reasonable approximation of the percentage of Kv1.5 channels inactivated by Kvbeta 1.3. Increases in the ratio of beta - to alpha -subunit cRNA failed to further enhance inactivation and often suppressed current. This suppression occurred with both the wild-type and mutant beta -subunits and is in agreement with the data of Accili et. al. (1) showing that overexpression of the conserved Kvbeta 1 core can decrease Kv1.5 current.

Because deletion of the amino terminus of Shaker also prevents fast inactivation (19, 20) and because transfer of that domain to a delayed rectifier channel confers N-type inactivation (21, 38), we made two tandem constructs to determine whether the amino-terminal domain of Kvbeta 1.3 also constituted an independent inactivation domain. Expression of the tandem(N19) construct resulted in currents that were macroscopically indistinguishable from wild-type Kv1.5 (data not shown). However, as shown in Fig. 2D, the construct in which nearly all of the variable domain of the amino terminus of the beta -subunit was added, tandem(N87), reconstituted the hallmark of Kvbeta 1.3-mediated inactivation: at more positive voltages the current displayed both rapid and slow components of inactivation, whereas at voltages negative to +10 mV no apparent inactivation was observed. The rate of recovery from fast inactivation for tandem(N87) was too fast for reliable quantitation (time constant < 5 ms). This result localizes the functional domain responsible for fast and slow inactivation to the amino terminus of Kvbeta 1.3 and suggests that the first 19 amino acids do not constitute a complete inactivation particle. These data also indicate that the partial inactivation shown in Fig. 2B is unlikely to result from suboptimal levels of beta -subunit expression.

The hyperpolarizing shift in the midpoint of activation does not require an intact amino terminus. Figure 3 shows activation curves constructed from normalized deactivating tail current magnitudes as described in MATERIALS AND METHODS. Coexpression of Kv1.5 with the wild-type Kvbeta 1.3 subunit produced the greatest leftward shift compared with Kv1.5 expressed alone. Coexpression of Kv1.5 with Kvbeta 1.3(Delta N10) also shifted the curve leftward but to a slightly lesser extent. These data demonstrate the functional independence of the Kvbeta 1.3-mediated effects on inactivation and the change in the midpoint of activation. As shown in Fig. 3, the 68-amino acid deletion still caused a shift in the activation curve. The Kvbeta 1.3(Delta N91) construct did not induce a shift in the voltage dependence of activation (data not shown). Neither tandem construct (N19 or N87) shifted the midpoint of activation relative to wild-type Kv1.5 (Table 1). All of these data suggest that the beta -subunit domain involved in inactivation is distinct from that governing the shift in the voltage dependence of activation.


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Fig. 3.   Hyperpolarizing shift in midpoint of activation does not require an intact amino terminus. Activation curves obtained from oocytes expressing Kv1.5 in absence or presence of Kvbeta 1.3, Kvbeta 1.3(Delta N10), or Kvbeta 1.3(Delta N68) are compared with that obtained from expression of tandem(N87). Curves were created from Boltzmann equations fit to normalized deactivating tail current magnitudes as described in MATERIALS AND METHODS.

Prolonged depolarization revealed a slow component of inactivation. Fast inactivation occurred with a time constant of 5.7 ms, suggesting that this process is essentially at steady state within 25 ms (Table 1 and Fig. 4B, inset). However, the current continued to decay at a slower rate after this time, indicating a slow component of inactivation. To examine the slow component of inactivation, oocytes expressing Kv1.5 or Kv1.5 + Kvbeta 1.3 were depolarized for 5 s. Unscaled currents resulting from 5-s depolarizations to +70, +50, and +30 mV are shown in Fig. 4, A and B, for Kv1.5 and Kv1.5 + Kvbeta 1.3, respectively. The magnitude of current measured at 1 s increases with membrane potential in the absence of Kvbeta 1.3 (Fig. 4A). However, in the presence of Kvbeta 1.3, the K+ current measured at 1 s decreases with depolarizations positive to +30 mV (Fig. 4B). Because fast inactivation is complete within 50 ms (Fig. 4B, inset), normalization to the current magnitude at 100 ms allows qualitative comparison of slow inactivation in the absence and presence of Kvbeta 1.3 (Fig. 4, C and D). The normalized tracings in the absence of Kvbeta 1.3 superimposed (Fig. 4C), indicating that the extent and rate of slow inactivation of Kv1.5 was independent of membrane potential, i.e., the tracings reach a plateau at -10 mV (36). In contrast, the tracings at each membrane potential in the presence of Kvbeta 1.3 did not superimpose (Fig. 4D), indicating that the extent of slow inactivation depends on the membrane potential.


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Fig. 4.   Kvbeta 1.3 enhances slow inactivation through a mechanism that requires a functional fast inactivation domain. Oocytes expressing Kv1.5 (A and C) or Kv1.5 + Kvbeta 1.3 (B and D) were depolarized for 5 s to +70, +50, and +30 mV. To emphasize differences in slow decay rates, currents were normalized to amplitude at 100 ms (indicated by arrows in A and B) and superimposed as shown in C and D, respectively. E: experimental pulse protocol used to quantitate slow component of inactivation and a typical current tracing from an oocyte expressing Kv1.5 + Kvbeta 1.3. Slow inactivation (S) was defined as amount of current that did not recover within 50 ms at -80 mV, fast inactivation (F) as amount that recovered during 50-ms pulse, and open (O) as noninactivated current. F: data obtained from a minimum of 4 oocytes expressing Kv1.5, Kv1.5 + Kvbeta 1.3, Kv1.5+ Kvbeta 1.3(Delta N10), and tandem(N87) were averaged and plotted as fraction of slow inactivation vs. duration of 1st depolarizing pulse. Error bars, SE. Average values were fit with a single exponential. P1 and P2, peak currents of 1st and 2nd pulses, respectively.

To further quantitate the extent of slow inactivation, oocytes expressing Kv1.5, Kv1.5 + Kvbeta 1.3, Kv1.5 + Kvbeta 1.3(Delta N10), or tandem(N87) were studied using a two-pulse protocol, as illustrated in Fig. 4E. Figure 4E also shows a representative tracing from an oocyte injected with Kv1.5 + Kvbeta 1.3 cRNAs. Because the functional effects were similar for each amino-terminal deletion, we fully characterized only Kvbeta 1.3(Delta N10). The following operational definitions of fast and slow inactivation were used: slow inactivated channels were defined as being unable to recover within 50 ms at -80 mV, whereas fast inactivated channels fully recover within this interval. In Fig. 4F, average values for the fraction of slow inactivation are plotted as a function of the duration of the first pulse. The extent of slow inactivation at 500 ms observed in the presence of the wild-type Kvbeta 1.3 subunit is fourfold greater than that observed with Kv1.5 alone (0.2 ± 0.02, n = 5, and 0.05 ± 0.004, n = 5, respectively). No increase in slow inactivation was observed in mutants lacking fast inactivation [Kvbeta 1.3(Delta N10)]. The extent of both fast and slow inactivation observed with the tandem(N87) construct was less than those observed in presence of the wild-type beta -subunit, suggesting a correlation between the extent of fast inactivation and the enhancement of slow inactivation.

Characterization of Kvbeta 1.3 interaction with internal and external Kv1.5 pore mutations. One possible mechanism for the fast component of inactivation is that the amino terminus of Kvbeta 1.3 acts as an open channel blocker that binds within the open pore. Because the antiarrhythmic agent quinidine probably interacts by such a mechanism (44), we tested whether a Kv1.5 point mutant that affects quinidine block (V512A) would also affect Kvbeta 1.3-mediated inactivation. The Kv1.5(V512A) mutation is located in the internal mouth of the ion-conducting pore and has three major effects on Kv1.5 function (44): a shift in the midpoint of activation (Table 1), a slowing of deactivation, and a fourfold increase in the affinity for quinidine. If the binding site for the inactivation particle of Kvbeta 1.3 overlaps that of quinidine, then the V512A mutation may also show an enhanced affinity for the beta -subunit blocking particle (and hence display more inactivation). Currents elicited from oocytes expressing the Kv1.5(V512A) mutant alpha -subunit in the absence and presence of Kvbeta 1.3 are shown in Fig. 5, A and B. The internal pore mutation (V512A) did not affect either the Kvbeta 1.3-mediated inactivation or the hyperpolarizing shift in the midpoint of activation (Fig. 5 and Table 1). Another internal pore mutation of Kv1.5, T505I, which enhances quinidine block ~10-fold (44), also did not affect Kvbeta 1.3 function (data not shown).


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Fig. 5.   An external pore mutation alters fast inactivation. Current from oocytes expressing Kv1.5 subunit point mutants V512A (A and B) and R485Y (C and D) are shown in absence (A and C) and presence (B and D) of Kvbeta 1.3. Currents in A and B were elicited by 200-ms depolarizations from a holding potential of -100 mV to potentials between -90 and +70 mV in 10-mV increments. Currents in C and D were elicited by 200-ms depolarizations from a holding potential of -80 mV to potentials between -60 and +70 mV in 10-mV increments. Test potentials were followed by a repolarizing step to -40 mV to record deactivating currents.

In contrast to the internal pore point mutations, which do not alter beta -subunit-mediated effects, an external pore mutation (R485Y) dramatically increased the extent of beta -induced fast inactivation (Fig. 5D and Table 1). This mutation also reduces C-type inactivation (23, 24, 44) and renders the channel sensitive to external tetraethylammonium (TEA; Ref. 25 and T. C. Rich and D. J. Snyders, unpublished results). In addition, currents from oocytes expressing Kv1.5 (R485Y) did not show significant slow inactivation after 100 ms in either the absence or presence of Kvbeta 1.3. The R485Y mutation also increased the Kvbeta 1.3-mediated prolongation of the deactivating tail current at -40 mV (2.4-fold for Kv1.5 vs. 3.9-fold for R485Y; Table 1). The deactivating tail current magnitude of Kv1.5(R485Y) + Kvbeta 1.3 at -40 mV exceeded the steady-state current magnitude of depolarizing pulses from +40 mV to +70 mV, despite a difference of up to 110 mV in the driving force. This transition was complete within 6 ms of the membrane potential change, indicating that recovery (unblock) from the enhanced fast inactivation is extremely rapid. The R485Y mutation in Kv1.5 did not alter the shift in the midpoint of activation or the time constant of the fast component of inactivation (Table 1). In contrast to the effects on Kvbeta 1.3-mediated inactivation, this external pore mutation does not alter the action of open channel-blocking drugs (44), indicating that Kvbeta 1.3-mediated fast inactivation is not influenced by the same alpha -subunit amino acid residues that are involved in quinidine block.

Effect of high external K+ on inactivation. Sensitivity of block to concentrations of the conducting ion has been used to support an open pore model of block by both pore-blocking drugs and the inactivation particle (11). Increasing the external K+ concentration also reduces the slow component of inactivation (2, 24, 31). To test whether Kvbeta 1.3-mediated inactivation was sensitive to external K+ concentration, we compared the Kvbeta 1.3-mediated fast and slow inactivation of Kv1.5 and Kv1.5(R485Y) in the absence and presence of 96 mM extracellular K+ (Fig. 6). High external K+ concentrations reduced the extent of both fast and slow inactivation. Figure 6, A and B, shows representative current tracings during depolarizations to +70 mV in oocytes expressing Kv1.5 + Kvbeta 1.3 and Kv1.5 (R485Y) + Kvbeta 1.3, either in normal K+, in high K+, or after return to normal K+ (wash). Currents were normalized to the peak to emphasize the difference in the extent of inactivation. For Kvbeta 1.3 with either the wild-type or R485Y alpha -subunit, the presence of high external K+ reduced the extent of inactivation at 200 ms (Table 1). To compare the time course of inactivation, currents from Fig. 6, A and B, were further normalized to the current level at 1 s as shown in Fig. 6, C and D. For the wild-type subunit, increasing external K+ eliminated the slow component of inactivation without affecting the rate of fast inactivation (see also Table 1). The slow component of inactivation returned on washout of external K+. Figure 6D illustrates that the rate of slow inactivation of Kv1.5(R485Y) in the presence of Kvbeta 1.3 was not affected by the presence of 96 mM K+.


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Fig. 6.   High external K+ reduces extent of fast inactivation. Oocytes expressing Kv1.5 + Kvbeta 1.3 (A and C) or Kv1.5 (R485Y) + Kvbeta 1.3 (B and D) were depolarized to +70 mV for 1 s, and currents were recorded in normal K+ (2 mM KCl), high K+ (96 mM KCl) and after washing back to normal K+. A and B: currents were normalized to peak to compare extent of inactivation. For each tracing, slow inactivation continued at same rate for duration of 1-s pulse (only 1st 300 ms are shown). C and D: currents were further scaled to steady-state current at 1 s to compare inactivation rates under different conditions. Again, 1st 300 ms after peak are shown.

Voltage dependence of the inactivation induced by Kvbeta 1.3. Two lines of evidence suggested that Kvbeta 1.3-mediated inactivation was voltage dependent. First, macroscopic inactivation was apparent only at large depolarizations. Second, the steady-state current level from depolarizations more positive than +20 mV does not increase with a corresponding increase in the membrane potential (12), which would occur if the open channel probability were decreasing as the driving force increased. To quantitate this voltage dependence, we used a two-pulse protocol in which the membrane potential of the first pulse was varied as shown in Fig. 7A. Figure 7B shows normalized data plotted vs. the membrane potential of the 25-ms first pulse. Because fast inactivation is essentially at steady state and slow inactivation is negligible at 25 ms, this approach isolates the fast component of inactivation. Data were obtained in both the absence and presence of 96 mM extracellular K+. The fractional inactivation increased rapidly in the voltage range corresponding to channel activation, indicating that Kvbeta 1.3-mediated inactivation required the channel to enter the open state. Note that the -10 mV tracing in Fig. 7A shows no apparent inactivation in the first pulse, whereas there was an obvious reduction in the peak current of the second pulse, i.e., channels inactivate at -10 mV even though inactivation is not apparent in the macroscopic current. Positive to potentials at which the channels are maximally activated (+10 mV), the fractional inactivation continued to increase with membrane potential, indicating that Kvbeta 1.3-mediated fast inactivation was voltage dependent (Fig. 7B). The Woodhull model (42) was used to quantitate the voltage dependence, and the derived z · delta  values are indicated in Fig. 7. Similar curves and z · delta  values were obtained with tandem(N87) and the alpha -subunit point mutants (Fig. 7C). These data suggest that the voltage dependence of Kvbeta 1.3-mediated block is independent of individual alpha -subunit properties, consistent with the localization of the voltage-dependent inactivating particle to the amino terminus of Kvbeta 1.3 (Fig. 2). The fits are extrapolated to negative membrane potentials to predict the fractional block of open channels at those voltages. Note that elevating external K+ to 96 mM reduces the extent of fast inactivation but does not alter its voltage dependence (z · delta ).


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Fig. 7.   Fast inactivation is mediated by external K+-sensitive, voltage-dependent, open channel block. Voltage dependence of inactivation was quantitated using a 2-pulse protocol as depicted in A. In 1st pulse, oocytes were depolarized to varying potentials for either 25 ms (A-C) or 1 s (D) and stepped to +70 mV for 200 ms in 2nd pulse. A: tracings from test pulses of -70, -10, and +70 mV. Time point of peak current of 2nd pulse was determined from a 1st pulse potential of -70 mV and is represented by an arrow. Current amplitudes of subsequent pulses were determined at this time point, normalized with respect to maximum, and plotted vs. potential of 1st depolarizing pulse (B). Similar plots were made for tandem(N87) and alpha -subunit point mutants (C). A Boltzmann equation was fit to data from voltages exceeding that of maximal activation (greater than +20 mV). Fits (solid lines) are extended into negative voltages to predict fractional inactivation of open channels at those voltages. Using the Woodhull model (42), z · delta  values were determined from derived slope factor (z represents effective charge of blocking particle, and delta  represents fraction of transmembrane potential at blocking site). Data obtained in high external K+ (96 mM) are indicated (B and D). Data are from 5 oocytes.

To examine the voltage dependence of slow inactivation, we extended the duration of the first pulse to 1 s (Fig. 7D). The 1-s first pulse reveals the combined voltage dependence of the fast and slow components of inactivation. From Fig. 7D, the voltage dependence (z · delta ) of inactivation at 1 s is greater than that at 25 ms (fast alone), suggesting that the slow component of inactivation contributes to the observed voltage dependence.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Separation of Kvbeta 1.3-mediated functional effects. Other investigators have suggested that the hyperpolarizing shift in the activation curve observed following coexpression of Kv1.5 with Kvbeta 1.2 is specifically linked to inactivation and that the beta -subunit does not directly alter activation properties (10). Precise quantitation of the voltage dependence of activation is difficult in the presence of the voltage-dependent inactivation observed with open channel block. Once current values are scaled, an artifactual shift in the activation curve can be generated. Because analysis of peak current levels is especially prone to artifact, activation curves were obtained from the tail current amplitude 6 ms after repolarization to -40 mV. Because the time constant for recovery from fast inactivation at -80 mV was 5.1 ± 0.1 ms, these tail current measurements are not perfect. However, even before the experiments reported here with the Kvbeta 1.3 mutants, it seemed likely that, at least in the case of Kv1.5 and Kvbeta 1.3, the beta -subunit-induced activation shift was independent of inactivation. Indeed, current was observed at membrane potentials as low as -30 mV, a potential generating no current in the absence of the beta -subunit. In addition, the Kvbeta 2.1 subunit shifts the Kv1.5 activation curve without inducing fast inactivation (16, 40).

Fast inactivation, but not the Kvbeta 1.3-induced shift in the midpoint of activation, required the amino terminus of Kvbeta 1.3. A deletion of as few as 10 amino acids from the amino terminus of Kvbeta 1.3 prevented inactivation, whereas deletions of up to 68 amino acids retained the ability to hyperpolarize the midpoint of activation. However, the magnitude of the hyperpolarizing shift was reduced, as illustrated in Fig. 3, suggesting that a fraction of the shift observed with the wild-type Kvbeta 1.3 subunit was due to an imperfect activation curve. The Kvbeta 1.3(Delta N91) construct neither induced inactivation nor shifted the midpoint of activation, suggesting that the region between amino acids 68 and 91 may be important for the shift in the activation threshold. However, because Kvbeta 1.3(Delta N91) conferred no functional effects, we were unable to confirm synthesis of this truncated subunit or its association with Kv1.5 in the oocyte. Nevertheless, other investigators have shown that the Kvbeta 1 carboxy terminus assembles with Kv1.5 without altering channel kinetics (1). Together with the finding that addition of the first 87 amino acids reconstituted the voltage-dependent rapid inactivation but did not alter the midpoint of activation, these data indicate that distinct domains of Kvbeta 1.3 are responsible for hyperpolarizing the midpoint of activation and conferring voltage-dependent fast inactivation.

Deactivation is affected by fast inactivation. Prolonged deactivation has been observed in the presence of open pore-blocking drugs and N-type inactivation (11, 35, 37). Blockade of the pore by either the drug or inactivation particle is thought to prevent deactivation (11, 35). Thus the drug or inactivating particle must dissociate before the channel can close. The fact that the open channel is subject to reblock further complicates the deactivating current. An open channel will conduct current until it deactivates or is reblocked. If the channel becomes reblocked, deactivation is again prevented, and the particle must dissociate before the channel can deactivate. Therefore, reblock by the drug or inactivating particle can significantly contribute to prolonging the deactivating current (11). Shifting the midpoint of activation also results in altered kinetics of deactivation (39). The tandem(N87) construct, which conferred fast inactivation without altering the midpoint of activation, and the Kvbeta 1.3(Delta N10) truncation, which hyperpolarized the midpoint of activation without inducing fast inactivation, demonstrated deactivation time constants intermediate between those observed with Kv1.5 alone and Kv1.5 plus the wild-type Kvbeta 1.3. These data indicate that the prolonged deactivation observed in the wild-type subunit may be the result of the combined effects of a shift in the midpoint of activation and reentry into the inactivated state.

External, not internal, Kv1.5 pore mutations alter Kvbeta 1.3-induced inactivation. It was conceivable that the inactivation particle of Kvbeta 1.3 interacts with the alpha -subunit at the same cytoplasmic binding site as quinidine, since internal TEA derivatives can compete with N-type inactivation in Shaker (7). However, the two alpha -subunit point mutants, V512A and T505I, which increase quinidine block (44), failed to alter Kvbeta 1.3-induced inactivation. Thus the molecular determinants in Kv1.5 for Kvbeta 1.3-induced fast inactivation and open channel drug block appear to be distinct. However, it is still possible that there is overlap in the alpha -subunit domains involved in quinidine block and Kvbeta 1.3-induced inactivation.

The external pore mutation R485Y was used to determine whether the Kvbeta 1.3-mediated slow inactivation differed from the slow inactivation observed in the absence of the beta -subunit. This tyrosine substitution in the outer pore reduces slow inactivation in Kv1.5 (44) and other Shaker-like channels (24) and renders these channels sensitive to external TEA (Refs. 15, 25, Rich and Snyders, unpublished results). This point mutant prevented the Kvbeta 1.3-induced slow component of inactivation (Figs. 4 and 6, B and D), suggesting that Kvbeta 1.3 enhances the native Kv1.5 slow inactivation by a mechanism similar to that of internal drug block (2, 3). Therefore, the induced slow inactivation is likely a property of the individual alpha -subunit and not one separately conferred by Kvbeta 1.3. The R485Y point mutation also unexpectedly increased the extent of both fast inactivation and the prolongation of the deactivating tail current, without affecting either the rate and voltage dependence of fast inactivation or the hyperpolarizing shift in the midpoint of activation. This selective enhancement of fast inactivation further supports the conclusion that Kvbeta 1.3 effects on activation and inactivation are functionally independent. The slow deactivation again supports the idea that deactivation rate is directly modified by fast inactivation.

Wang et al. (41) have expressed Kvbeta 1.3 with several different Kv1 subfamily alpha -subunit isoforms and reported variable effects on the expressed currents. Comparison of the amino acids in the internal mouth of the pore reveals only two variable amino acids, neither of which correlates with the observed differences. Included in the analysis was Kv1.1, which has a tyrosine in the equivalent position of Arg-485. Kvbeta 1.3 conferred only 10% inactivation on Kv1.1 at 100 ms of a +60 mV pulse (41). In contrast to the quaternary ammonium derivatives (8, 25, 37), it is unlikely that a small number of amino acids at the internal or external pore will determine sensitivity to Kvbeta 1.3-mediated fast inactivation.

Inactivation is dependent on both external K+ and voltage. A model introduced by Baukrowitz and Yellen (2-4) for the Shaker channel proposes that occupancy of an external K+ binding site inhibits slow inactivation. This model proposes that the enhancement of slow inactivation in the presence of internal blockade (drug or inactivation ball) is a direct result of depletion of K+ from this external site. Increasing the external K+ concentration allows occupancy of this site, despite internal blockade, returning the extent of slow inactivation to the level observed in the absence of the blocking agent. In agreement with this model, elevating external K+ inhibited the slow component of inactivation, as seen in Fig. 6C. However, it also clearly inhibited the extent of fast inactivation, as shown in Figs. 6B and 7B. Because the extent of slow inactivation appears to be coupled to the extent of fast inactivation (Fig. 4F), we cannot discern whether the reduction in slow inactivation is the result of reduced fast inactivation or a direct effect of K+ on slow inactivation.

The voltage dependence of Kvbeta 1.3-mediated fast inactivation contrasts with that of N-type inactivation in Shaker and the fast inactivation conferred on Kv1.5 by Kvbeta 1.2, both of which are insensitive to membrane potential (10, 46). The voltage dependence observed with Kvbeta 1.3 may be due to charge within the blocking particle sensing the transmembrane potential, as predicted by the Woodhull model (42). Alternatively, the voltage dependence could reside in voltage-sensitive conformations in the alpha -subunit itself. The present data cannot distinguish between these two possibilities.

In conclusion, previous characterization of the Kvbeta 1.3 subunit demonstrated several modifications of Kv1.5 currents, including 1) a rapid, but incomplete, inactivation that is only apparent at large depolarizations, 2) a 13-mV hyperpolarizing shift in the midpoint of activation, and 3) a 2.4-fold slowing of deactivation (12). Here, we show that these diverse functional effects are the result of interactions of at least two different domains of the Kvbeta 1.3 subunit with the Kv1.5 alpha -subunit. The Kvbeta 1.3-mediated fast inactivation occurred by an open channel block mechanism that was sensitive to both external K+ and membrane potential, and this fast inactivation induced a slow component of inactivation. The molecular determinants for quinidine and quaternary ammonium pore block are distinct from those involved in Kvbeta 1.3-mediated inactivation. Furthermore, an external pore mutation that governs external TEA sensitivity and slow inactivation allosterically enhances Kvbeta 1.3-mediated fast inactivation.

    ACKNOWLEDGEMENTS

We thank Brady Palmer and Michelle Choi for technical assistance in oocyte preparation and injection.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-49330 (to M. M. Tamkun), HL-47599 (to D. J. Snyders), and HL-46681 (to D. J. Snyders, P. B. Bennett, and M. M. Tamkun) and by a United Negro College Fund-Merck Postdoctoral Science Research Fellowship (to S. K. England).

P. B. Bennett is an Established Investigator of the American Heart Association.

Present addresses: S. K. England, Dept. of Physiology and Biophysics, University of Iowa School of Medicine, Iowa City, IA 52242-1109; V. N. Uebele, Merck Research Labs, WP26-265, West Point, PA 19486.

Address for reprint requests: M. M. Tamkun, Dept. of Physiology, Colorado State University, Fort Collins, CO 80523.

Received 18 December 1997; accepted in final form 17 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Accili, E. A., J. Kiehn, Q. Yang, Z. G. Wang, A. M. Brown, and B. A. Wible. Separable Kv beta subunit domains alter expression and gating of potassium channels. J. Biol. Chem. 272: 25824-25831, 1997[Abstract/Free Full Text].

2.   Baukrowitz, T., and G. Yellen. Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron 15: 951-960, 1995[Medline].

3.   Baukrowitz, T., and G. Yellen. Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K+ channels. Proc. Natl. Acad. Sci. USA 93: 13357-13361, 1996[Abstract/Free Full Text].

4.   Baukrowitz, T., and G. Yellen. Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K+ channel. Science 271: 653-656, 1996[Abstract].

5.  Catterall, W., and P. N. Epstein. Ion channels. Diabetologia 35, Suppl. 2: S23-S33, 1992.

6.   Chandy, K. G., and G. A. Gutman. Voltage-gated K+ channels. In: Handbook of Receptors and Channels: Ligand- and Voltage-Gated Ion Channels, edited by R. A. North. Boca Raton, FL: CRC, 1995, p. 1-71.

7.   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].

8.   Choi, K. L., C. Mossman, J. Aube, and G. Yellen. The internal quaternary ammonium receptor site of Shaker potassium channels. Neuron 10: 533-541, 1993[Medline].

9.   Chouinard, S. W., G. F. Wilson, A. K. Schlimgen, and B. Ganetzky. A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus. Proc. Natl. Acad. Sci. USA 92: 6763-6767, 1995[Abstract].

10.   De Biasi, M., Z. Wang, E. Accili, B. Wible, and D. Fedida. Open channel block of human heart hKv1.5 by the beta -subunit hKvbeta 1.2. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H2932-H2941, 1997[Abstract/Free Full Text].

11.   Demo, S. D., and G. Yellen. The inactivation gate of the Shaker K+ channel behaves like an open-channel blocker. Neuron 7: 743-753, 1991[Medline].

12.   England, S. K., V. N. Uebele, J. Kodali, P. B. Bennett, and M. M. Tamkun. A novel K+ channel beta-subunit (hKv beta 1.3) is produced via alternative mRNA splicings. J. Biol. Chem. 270: 28531-28534, 1995[Abstract/Free Full Text].

13.   England, S. K., V. N. Uebele, H. Shear, J. Kodali, P. B. Bennett, and M. M. Tamkun. Characterization of a novel K+ channel beta subunit expressed in human heart. Proc. Natl. Acad. Sci. USA 92: 6309-6313, 1995[Abstract].

14.   Fink, M., F. Duprat, F. Lesage, C. Heurteaux, G. Romey, J. Barhanin, and M. Lazdunski. A new K+ channel beta subunit to specifically enhance Kv2.2 (CDRK) expression. J. Biol. Chem. 271: 26341-26348, 1996[Abstract/Free Full Text].

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

16.   Heinemann, S. H., J. Rettig, H. R. Graack, and O. Pongs. Functional characterization of Kv channel beta-subunits from rat brain. J. Physiol. (Lond.) 493: 625-633, 1996[Abstract].

17.   Heinemann, S. H., J. Rettig, F. Wunder, and O. Pongs. Molecular and functional characterization of a rat brain K-v beta 3 potassium channel subunit. FEBS Lett. 377: 383-389, 1995[Medline].

18.   Hille, B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer, 1992.

19.   Hoshi, T., W. N. Zagotta, and R. W. Aldrich. Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region. Neuron 250: 568-571, 1991.

20.   Hoshi, T., W. N. Zagotta, and R. W. Aldrich. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250: 533-538, 1990[Medline].

21.   Iverson, L. E., and B. Rudy. The role of the divergent amino and carboxyl domains on the inactivation properties of potassium channels derived from the Shaker gene of Drosophila. J. Neurosci. 10: 2903-2916, 1990[Abstract].

22.   Lewis, R. S., and M. D. Cahalan. Potassium and calcium channels in lymphocytes. Annu. Rev. Immunol. 13: 623-653, 1995[Medline].

23.   Liu, Y., M. E. Jurman, and G. Yellen. Dynamic rearrangement of the outer mouth of a K+ channel during gating. Neuron 16: 859-867, 1996[Medline].

24.   Lopez-Barneo, J., T. Hoshi, S. H. Heinemann, and R. W. Aldrich. Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Receptors Channels 1: 61-71, 1993[Medline].

25.   MacKinnon, R., and G. Yellen. Mutations affecting TEA blockade and ion permeation in voltage-activated K+ channels. Science 250: 276-279, 1990[Medline].

26.   Majumder, K., M. Debiasi, Z. G. Wang, and B. A. Wible. Molecular cloning and functional expression of a novel potassium channel beta-subunit from human atrium. FEBS Lett. 361: 13-16, 1995[Medline].

27.   McCormack, K., T. McCormack, M. Tanouye, B. Rudy, and W. Stuhmer. Alternative splicing of the human Shaker K+ channel beta 1 gene and functional expression of the beta 2 gene product. FEBS Lett. 370: 32-36, 1995[Medline].

28.   Morales, M. J., R. C. Castellino, A. L. Crews, R. L. Rasmusson, and H. C. Strauss. A novel beta subunit increases rate of inactivation of specific voltage-gated potassium channel alpha subunits. J. Biol. Chem. 270: 6272-6277, 1995[Abstract/Free Full Text].

29.   Morales, M. J., J. O. Wee, S. M. Wang, H. C. Strauss, and R. L. Rasmusson. The N-terminal domain of a K+ channel beta subunit increases the rate of C-type inactivation from the cytoplasmic side of the channel. Proc. Natl. Acad. Sci. USA 93: 15119-15123, 1996[Abstract/Free Full Text].

30.   Nakahira, K., G. Y. Shi, K. J. Rhodes, and J. S. Trimmer. Selective interaction of voltage-gated K+ channel beta-subunits with alpha-subunits. J. Biol. Chem. 271: 7084-7089, 1996[Abstract/Free Full Text].

31.   Pardo, L. A., S. H. Heinemann, H. Terlau, U. Ludewig, C. Lorra, O. Pongs, and W. Stuhmer. Extracellular K+ specifically modulates a rat brain K+ channel. Proc. Natl. Acad. Sci. USA 89: 2466-2470, 1992[Abstract].

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

33.   Scott, V. E., J. Rettig, D. N. Parcej, J. N. Keen, J. B. Findlay, O. Pongs, and J. O. Dolly. Primary structure of a beta subunit of alpha-dendrotoxin-sensitive K+ channels from bovine brain. Proc. Natl. Acad. Sci. USA 91: 1637-1641, 1994[Abstract].

34.   Sewing, S., J. Roeper, and O. Pongs. Kv beta 1 subunit binding specific for shaker-related potassium channel alpha subunits. Neuron 16: 455-463, 1996[Medline].

35.   Snyders, D. J., K. M. Knoth, S. L. Roberds, and M. M. Tamkun. Time-, state- and voltage-dependent block by quinidine of a cloned human cardiac channel. Mol. Pharmacol. 41: 332-339, 1992.

36.   Snyders, D. J., M. M. Tamkun, and P. B. Bennett. A rapidly activating and slowly inactivating K+ channel from human heart. Functional analysis after stable mammalian cell culture expression. J. Gen. Physiol. 101: 513-543, 1993[Abstract].

37.   Snyders, D. J., and S. W. Yeola. Determinants of antiarrhythmic drug action electrostatic and hydrophobic components of block of the human cardiac hKv1.5 channel. Circ. Res. 77: 575-583, 1995[Abstract/Free Full Text].

38.   Stocker, M., O. Pongs, M. Hoth, S. H. Heinemann, W. Stuhmer, K. H. Schroter, and J. P. Ruppersberg. Swapping of functional domains in voltage-gated K+ channels. Proc. Natl. Acad. Sci. USA 245: 101-107, 1991.

39.   Tytgat, J., and P. Daenens. Effect of lanthanum on voltage-dependent gating of a cloned mammalian neuronal potassium channel. Brain Res. 749: 232-237, 1997[Medline].

40.   Uebele, V. N., S. K. England, A. Chaudhary, M. M. Tamkun, and D. J. Snyders. Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kv beta 2.1 subunits. J. Biol. Chem. 271: 2406-2412, 1996[Abstract/Free Full Text].

41.   Wang, Z. G., J. Kiehn, Q. Yang, A. M. Brown, and B. A. Wible. Comparison of binding and block produced by alternatively spliced Kv beta 1 subunits. J. Biol. Chem. 271: 28311-28317, 1996[Abstract/Free Full Text].

42.   Woodhull, A. M. Ionic blockade of sodium channels in nerve. J. Gen. Physiol. 61: 2316-2325, 1973.

43.   Xu, J., W. F. Yu, Y. N. Jan, L. Y. Jan, and M. Li. Assembly of voltage-gated potassium channels: conserved hydrophilic motifs determine subfamily-specific interactions between the alpha-subunits. J. Biol. Chem. 270: 24761-24768, 1995[Abstract/Free Full Text].

44.   Yeola, S. W., T. C. Rich, V. N. Uebele, M. M. Tamkun, and D. J. Snyders. Molecular analysis of a binding site for quinidine in a human cardiac delayed rectifier K+ channel: role of S6 in antiarrhythmic drug binding. Circ. Res. 78: 1105-1114, 1996[Abstract/Free Full Text].

45.   Yu, W. F., J. Xu, and M. Li. NAB domain is essential for the subunit assembly of both alpha-alpha and alpha-beta and complexes of Shaker-like potassium channels. Neuron 16: 441-453, 1996[Medline].

46.   Zagotta, W. N., T. Hoshi, and R. W. Aldrich. Gating of single Shaker potassium channels in Drosophila muscle and in Xenopus oocytes injected with Shaker mRNA. Proc. Natl. Acad. Sci. USA 86: 7243-7247, 1989[Abstract].


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