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Separable effects of human Kv1.2 N- and C-termini on inactivation and expression of human Kv1.4

E. A. Accili, Y. A. Kuryshev *, B. A. Wible ¹ and A. M. Brown *

Received 19 January 1998; accepted after revision 6 July 1998.

	ABSTRACT

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1. The Kv subunits of voltage-gated K⁺ channels alter the functional expression and gating of non- or slowly inactivating Kv1 subunits via two separate domains. To determine how Kv subunits modulate a rapidly inactivating Kv1 subunit, we did two-microelectrode voltage clamp experiments on human Kv1.4 voltage-gated K⁺ channels expressed heterologously in Xenopus oocytes. In addition we tested a slowly inactivating mutant of Kv1.4 lacking amino acids 2-146 of the N-terminal -ball domain (Kv1.4N2-146). Kv1.4 or Kv1.4N2-146 were co-expressed with either rat Kv2 or human Kv1.2. To separate domain effects, we also used a mutant of Kv1.2 lacking the unique 79 amino acid N-terminal -ball domain (Kv1-C).

2. For the mutant Kv1.4N2-146 we found that Kv1-C or Kv2 increased current amplitude without altering activation or inactivation. By contrast Kv1.2 produced rapid inactivation and slowed deactivation due to block produced by the -ball. The -ball also increased the rate of C-type inactivation in 5 mM, but not 50 mM, external K⁺ consistent with an effect of blockade on K⁺ efflux.

3. For Kv1.4, Kv1-C produced a voltage-independent increase in the rate of inactivation and shifted the inactivation curve to more hyperpolarized potentials, but had no effect on deactivation. Kv1-C, Kv2 and Kv1.2 slowed recovery from inactivation similarly, thereby excluding involvement of the -ball. Kv1.2 produced an additional more rapid, voltage-dependent component of inactivation, significantly reduced peak outward current and shifted steady-state inactivation towards hyperpolarized potentials.

4. Yeast two-hybrid studies showed that - interaction was restricted to the N-terminus of Kv1.4 and the C-terminus of Kv1.2 or Kv2. Direct interaction with the -ball did not occur. Our interpretation is that Kv1-C and Kv2 enhanced N-type inactivation produced by the Kv1.4 -ball allosterically.

5. We propose that Kv1.2 has three effects on Kv1.4, the first two of which it shares with Kv2. First, Kv1-C and Kv2 have a current-enhancing effect. Second, Kv1-C and Kv2 increase block by the -ball allosterically. Third, the -ball of K1.2 directly blocks both Kv1.4 and Kv1.4N2-146. When both - and -balls are present, competition for their respective binding sites slows the block produced by either ball.

	INTRODUCTION

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Transient A-type K⁺ currents are important for regulating the firing frequency of neurons (Connor & Stevens, 1971) and for regulating action potential propagation in axons (Debanne et al. 1997). Molecular cloning, co-precipitation and co-localization experiments and current measurements suggest that hetero-oligomeric assemblies of Kv1.4 and modulatory Kv subunits may underlie inactivating potassium channels in central neurons in vivo (Shenget al. 1993; Rettig et al. 1994; Rhodes et al. 1995, 1997; Sewing et al. 1996; Yu et al. 1996; Shamotienko et al. 1997). Kv subunits have been shown to alter the kinetics (Heinemann et al. 1994; Rettig et al. 1994; Majumder et al. 1995; England et al. 1995; Morales et al. 1995; Wang et al. 1996; Heinemann et al. 1996; De Biasi et al. 1997; Accili et al. 1997a,b) and expression (Shi et al. 1996; Accili et al. 1997a) of non-inactivating Kv1 subunits. For Kv1.2, two separate domains are involved; the N-terminus ball domain (-ball) identified by its net positive charge (Rettig et al. 1994) was shown to be responsible for the kinetic effects as a result of open channel blockade (Wang et al. 1996; DeBiasi et al. 1997; Accili et al. 1997a) whereas the C-terminus was shown to be responsible for changes in functional expression (Accili et al. 1997a).

For the inactivating ferret Kv1.4 channel, Kv1.2 accelerated inactivation and slowed recovery from inactivation. For a slow C-type inactivating mutant of Kv1.4 lacking the positively charged -ball peptide (Castellino et al. 1995), Kv1.2 conferred rapid inactivation but did not alter recovery (Castellino et al. 1995), leading the authors to infer an interaction with the -ball. Kv2, which shares extensive amino acid homology with Kv1.2 in their C-termini, has been shown to accelerate inactivation of voltage-dependent Shaker K⁺ channels (McCormack et al. 1995) and rat Kv1.4 channels (McIntosh et al. 1997) but does not introduce rapid, N-type inactivation into non- or slowly inactivating Kv1 channels (Rettig et al. 1994). Taken together these results suggest that Kv subunits may alter inactivation by interacting directly with the -ball of Kv1.4.

In the present experiments we found that Kv1.2 C-terminus (Kv1-C) and Kv2 had enhancing effects on Kv1.4 currents. In addition, both increased inactivation of Kv1.4 but had no effects on the kinetics of Kv1.4N2-146, a mutant lacking the -ball. Both slowed recovery from inactivation of wild-type Kv1.4 in a manner identical to Kv1.2. Because our yeast two-hybrid studies showed that direct - interaction did not involve the -ball, we propose that the effects on inactivation are allosterically mediated. The -ball blocked Kv1.4 and the non-inactivating Kv1.4 mutant, and in the case of Kv1.4 competed with block produced by the -ball. The channel block by the -ball increased the rate of C-type inactivation of the Kv1.4 mutant in low (5 mM) K⁺ probably by inhibiting K⁺ efflux. For Kv2 the - interactions show that the C-terminus domain of Kvs enhances both expression and -ball blockade, suggesting a novel role in modulating transient K⁺ currents and hence the firing frequency of neurons in vivo.

	METHODS

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In vitro transcription of cRNAs and oocyte injection

Oocytes were isolated as previously described (Taglialatela et al. 1995). Stage V-VI Xenopus oocytes were surgically removed from anaesthetized frogs (0·2 % tricaine, 30 min) and defolliculation was achieved by exposing the oocytes to collagenase (2 mg ml^-1, 40 min) in solution (mM): 82·5 NaCl, 2·5 KCl, 1 MgCl₂, 5 Hepes and 100 µg ml^-1 gentamicin, pH 7·6. After removal of the ovarian lobes, the anaesthetized frogs were killed. Following this, oocytes were incubated at 19°C in (mM): 100 NaCl, 2 KCl, 1·8 CaCl₂, 1 MgCl₂, 5 Hepes, 2·5 pyruvic acid and 100 µg ml^-1 gentamicin, pH 7·6. One day after isolation, defolliculated oocytes were injected with cRNAs. cRNAs encoding Kv1.2, Kv1-C, Kv2, Kv1.4N2-146 and Kv1.4 were prepared as previously described (Wang et al. 1996; Accili et al. 1997a). cRNA concentrations were estimated on denaturing agarose gels stained with ethidium bromide by comparison with RNA standards. cRNAs were dissolved in 0·1 M KCl, stored at -80°C, and diluted and mixed immediately prior to injection. Oocytes were injected with 46 nl of cRNA solutions in 0·1 M KCl. Final concentrations of cRNA were: 2 ng µl^-1 for Kv1.4N2-146, 5 or 10 ng µl^-1 for Kv1.4 and 250 ng µl^-1 for Kv1.2, Kv1-C or Kv2.

Electrophysiology

Measurement of Xenopus oocyte whole-cell currents was performed using the standard two-microelectrode voltage clamp technique as described previously (Wang et al. 1996; Accili et al. 1997a). Electrodes filled with 3 M KCl had resistances of 0·2-0·5 M when measured in the bath solution containing (mM): 50 KOH, 55 NaOH, 0·5 CaCl₂, 100 Mes, 2 MgCl₂ and 10 Hepes, pH 7·3. We used 50 mM K⁺ in bath solution to slow C-type inactivation and reduce its contribution to the inactivation of Kv1.4 during short pulses. In some experiments measurements were performed in solution containing 5 mM K⁺. In this case K⁺ was replaced with an equivalent concentration of Na⁺. Measurements were done 3-5 days after cRNA injection. For comparison of whole cell current amplitudes, all measurements were done 3 days after oocyte injection. All recordings were made at room temperature (20-22°C). Chemicals were purchased from Sigma.

Data acquisition and analyses were performed with pCLAMP software (Axon Instruments). Data were low pass filtered at 2 or 5 kHz before digitalization at 10 kHz. Data are reported as means ± S.E.M. Comparisons between two groups of oocytes were performed by Student's t test and means are considered to be significantly different when P < 0·05.

Yeast two-hybrid interaction

Protein-protein interactions were monitored with the yeast Matchmaker Two-Hybrid System from Clontech (Palo Alto, CA, USA). Full-length human Kv1.2 (amino acids 1-408), Kv1.2-N terminus (amino acids 1-79) and Kv1-C (the C-terminal 329 amino acids of the Kv1 subfamily) were subcloned into the DNA binding domain (BD) vector, pGBT9, and Kv1.4-N (amino acids 1-305) and a truncated version, Kv1.4-N (amino acids 147-305), were subcloned into the activation domain (AD) vector, pGAD424, as previously described (Wang et al. 1996). To subclone the partial fragment of Kv1.4-N containing the ball peptide (amino acids 1-176) into pGAD424, in frame EcoR Iand Sal Isites were incorporated into the 5' and 3' ends, respectively, by polymerase chain reaction (PCR). The PCR amplified construct was sequenced to confirm the correct reading frame for the yeast fusion and to check that no unwanted PCR mutations were introduced. Protein-protein interactions were tested in yeast host strain Y190 by co-transformation with pairs of BD and AD fusion constructs according to the manufacturer's protocol. Transformants were initially plated on media lacking tryptophan (-trp) and leucine (-leu) and grown for 2·5 days at 30°C. To facilitate comparison of interactions, a representative colony from each co-transformation was spotted onto another -trp/-leu plate and grown for 24 h at 30°C prior to assay for -galactosidase activity using a filter lift assay as specified in Clontech protocols. Development of blue colour within 8 h indicated activation of the reporter gene, lacZ, and was scored as a positive interaction between two fusion proteins.

	RESULTS

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Kv subunits without ball domains increase current expression of the non-inactivating Kv1.4 ball mutant

Previously we reported that the C-terminus of Kv1.2 (Kv1-C) increased Kv1.2 currents but decreased Kv1.5 currents (Accili et al. 1997a). We co-expressed Kv1-C with a Kv1.4 mutant lacking the ball peptide (Kv1.4N2-146) to determine the effects on current amplitude without the confounding effects of - and/or -ball inactivation. Significant increases in current (2·69 ± 0·29 times that of control in 5 batches of oocytes) were observed (Fig. 1A) although the increases were larger for Kv1.2 (Fig. 1B). Kv2, whose C-terminus is highly homologous to Kv1-C, also increased Kv1.4N2-146 currents (3·19 times that of control in 28 oocytes from 1 batch; Fig. 1C).

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Figure 1. Kv subunits increase functional expression of the non-inactivating Kv1.4 mutant, Kv1.4N2-146 A, whole cell currents were measured in Xenopus oocytes injected with cRNAs for Kv1.4N2-146 alone or together with Kv1-C (also equivalent to the C-terminus of Kv1.2). Oocytes were held at -80 mV and pulsed to +80 mV in 10 mV steps of 100 ms duration. B, bar plot of whole-cell currents measured in Kv1.4N2-146 or Kv1.2, alone or together with Kv1-C. Current amplitudes were measured at the end of the pulse to +70 mV. Values represent means ± S.E.M. * and ** indicate a significant difference from the value of current for Kv1.4N2-146 and Kv1.2, respectively (P < 0·05, t test). Numbers above bars represent the total number of oocytes in each group for a particular batch of oocytes. C, bar plot of whole cell currents measured in Xenopus oocytes injected with Kv1.4N2-146 alone and with Kv2 (* P < 0·05, t test).

Neither Kv1-C (Fig. 1A) nor Kv2 (data not shown) produced rapid inactivation of Kv1.4N2-146 currents during the 100 ms test pulses. Nor did either alter the steady-state activation: half-maximal activation (V_0·5) and voltage dependence (k)values were -30·9 ± 0·9 and 8·4 ± 0·7 mV for Kv1.4N2-146 alone (n = 6), -33·1 ± 1·1 and 7·9 ± 0·8 mV for Kv1.4N2-146 plus Kv1-C (n = 8) and -30·2 ± 1·0 and 8·4 ± 0·8 mV for Kv1.4N2-146 plus Kv2 (n = 8), or the rate of deactivation (data not shown). We also found that Kv2 did not alter slow, C-type inactivation of Kv1.4N2-146. Four-second voltage pulses to +40 mV produced slow inactivation of Kv1.4N2-146 having single exponential time constants of 2·84 ± 0·08 s (n = 6) without and 3·50 ± 0·13 s (n = 6) with Kv2. Recovery from inactivation for Kv1.4N2-146 without and with Kv2 was also similar: recovery time constants were 3·37 ± 0·19 (n = 6) and 3·44 ± 0·14 s (n = 6), respectively. Kv1.2 was reported not to alter recovery from inactivation of Kv1.4N2-146 currents (Castellino et al. 1995).

The -ball produces rapid inactivation of Kv1.4N2-146

Unlike Kv1-C (Fig. 1A), Kv1.2 produced rapid inactivation of Kv1.4N2-146 (compare Fig. 2A and B). Deactivation was also slowed (compare tails between Fig. 2D and E) and the steady-state activation curve was steeper and shifted to more negative potentials although the activation threshold was unchanged (Fig. 2C). The rates of rapid inactivation produced by Kv1.2 are voltage dependent in the range of 50 to 80 mV (Fig. 4C). A tail current protocol (Fig. 2D and E) established that the rate of activation was unchanged (Fig. 2D -F). It has been suggested that Kv1.2 modulation of C-type inactivation in the ferret Kv1.4N2-146 was sensitive to external K⁺ concentration (Morales et al. 1996). We found that inactivation of Kv1.4N2-146 in the presence of Kv1.2 consisted of two components (Fig. 3A), a rapid component attributable to block by the Kv1.2 N-terminal ball domain and a much slower component involving a C-type inactivation. In 50 mM external K⁺ the rate of C-type inactivation was not altered by Kv1.2. However, in 5 mM K⁺, C-type inactivation was faster in the presence of Kv1.2 (Fig. 3B). The fast component was insensitive to external K⁺ concentration. The increased rate of C-type inactivation at 5 mM external K⁺ is probably due to inhibition of K⁺ efflux by -ball blockade.

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Figure 2. The Kv1.2 N-terminal ball domain produces N-type inactivation of the Kv1.4N2-146 mutant Whole cell currents were measured in Xenopus oocytes injected with cRNA for Kv1.4N2-146 with (A) and without (B) Kv1.2. Oocytes were held at -80 mV and pulsed to +80 mV in 10 mV steps of 125 ms in duration. Kv1.2 produced inactivation and slowed deactivation. C, steady-state activation curves were generated from peak tail currents, at -80 mV normalized to the maximum and plotted against test potential. Data were fitted with the following Boltzmann function: I/Imax = [1 + exp((V0·5 - V)/k]-1,where I is the current, V is the test potential and k is the Boltzmann constant. and represent normalized values determined from tail currents of oocytes injected with Kv1.4N2-146 and Kv1.4N2-146 plus Kv1.2, respectively. Kv1.2 shifted the curve to more negative potentials (-9·9 mV) and increased the curve steepness (k) from 10·4 ± 1·1 mV (n = 6) to 7·3 ± 0·5 mV (n = 6). Note that the threshold for activation is similar in both situations. The values for half-maximal activation (V0·5) are -31·0 ± 1·8 mV (n = 6) and -40·9 ± 1·3 mV (n = 6). D and E, tail current protocol was used to compare activation kinetics of currents in oocytes injected with Kv1.4N2-146 + Kv1.2 (D) or Kv1.4N2-146 alone (E). Oocytes were held at -90 mV, pulsed to +120 mV for times ranging from 1 to 23 ms and repolarized to -120 mV (100 mM K+ in external solution). F, normalized tail currents were plotted as a function of depolarization time. Time constants of activation were 2·8 ± 0·4 (n = 3) and 2·5 ± 0·3 ms (n = 3) for Kv1.4N2-146 () and Kv1.4N2-146 + Kv1.2 (), respectively.

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Figure 3. Kv1.2 increases the rate of C-type inactivation at low but not high external K+ concentrations A, superimposition of normalized current traces for Kv1.4N2-146 alone and with Kv1.2 at test potential to +70 mV for 1·6 s from a holding potential of -80 mV. The C-type inactivation time constant for Kv1.4N2-146 alone was 1289 ms. In 50 mM K+, Kv1.2 produced a rapid inactivation but did not alter the rate of the slow C-type inactivation; time constants were 3·9 and 865 ms, respectively. B, the same oocytes and protocol as in A but in 5 mM external K+. C-type inactivation is significantly faster in the presence of Kv1.2; time constants were 1012 and 227 ms without and with Kv1.2, respectively. Note that the fast time constant was unchanged (4·0 ms). C, the same oocytes as in B but at a faster time scale.

Separable effects of Kv1.2 N- and C-termini on inactivation of Kv1.4

To this point, we have shown that the C-terminus of Kv1.2 enhanced Kv1.4N2-146 current as did Kv2 and that the N-terminus of Kv1.2 introduced rapid, N-type inactivation into Kv1.4N2-146. Next, we compared the effects of the Kv1.2 C-terminus and intact Kv1.2 on intact Kv1.4. Normalized current traces are compared in Fig. 4A. In these experiments short pulses were utilized and the extracellular solution contained 50 mM K⁺ to minimize the contribution of C-type inactivation. We found that both Kv1-C and Kv1.2 accelerated Kv1.4 N-type inactivation but did so in different ways. The inactivating currents produced by Kv1.4 alone or with Kv1-C were fitted with a single exponential function. The rate of N-type inactivation was roughly doubled in the presence of Kv1-C (, Fig. 4B). The rates of Kv1.4 inactivation either with or without Kv1-C did not vary with membrane potential in the range of 50 to 80 mV.

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Figure 4. Kv1.2 C- and N-termini produce distinct changes in Kv1.4 inactivation kinetics A, normalized whole cell currents measured in Xenopus oocytes injected with cRNAs for Kv1.4 alone or with Kv1.2 or Kv1-C. Oocytes were held at -80 mV, pulsed to +80 mV for 125 ms and repolarized to -80 mV. B, plot of inactivation time constants versus test potential. , slower second time constant of inactivating currents produced by injection of Kv1.4 with Kv1.2 (n = 5) fitted with a sum of two exponential functions. and , time constants of inactivating currents produced by injection of Kv1.4 alone (n = 2) and Kv1.4 with Kv1-C (n = 5), respectively, fitted with a single exponential function. C, a plot of inactivation time constants versus test potential. , faster first time constant of inactivating currents produced by injection of Kv1.4 with Kv1.2 (the second time constant is shown by in B). The fast time constant is voltage dependent, decreasing by almost 50 % from +50 to +80 mV whereas the slower time constant is not. For comparison the fast time constant of inactivating currents produced by injection of Kv1.4N2-146 with Kv1.2 is shown (; n = 5). D, whole cell currents measured in Xenopus oocytes injected with cRNAs for Kv1.4 alone or with Kv1-C or Kv1.2. Oocytes were held at -80 mV, pulsed to +70 mV for 125 ms and repolarized to -80 mV.

Co-expression with intact Kv1.2 produced further effects on the N-type inactivation of Kv1.4. Inactivation now had two rates and was fitted with a sum of two exponentials. The small (Fig. 4C; ) and large time constants (Fig. 4B; ) bracket the time constants associated with intact Kv1.4 with and without Kv1.2-C. Unlike the other time constants, the small time constant is voltage dependent. We attribute the smallest time constant to block by the -ball of Kv1.2 and the larger time constant to block by the -ball of Kv1.4 in the presence of -ball blockade. Note that the -ball time constant is larger in the presence of the -ball (Fig. 4C) and the -ball time constant is larger in the presence of the -ball (Fig. 4B, compare with ).

In addition to its effect on N-type inactivation, Kv1-C produced a significant increase in the peak current of Kv1.4 as it did for Kv1.4N2-146 (Fig. 4D). At a test potential of +70 mV peak currents were 11·30 ± 2·42 (n = 5) and 21·10 ± 2·08 µA (n = 5) for Kv1.4 alone and Kv1.4 plus Kv1-C, respectively. By contrast Kv1.2 decreased the peak current levels: 7·54 ± 0·71 µA (n = 5; Fig. 4D).

As shown in Fig. 5D, Kv1.2-C shifts the inactivation curve of Kv1.4 to more negative potentials. However, intact Kv1.2 produces rapid, N-type inactivation having two time constants (Fig. 4). Using the amplitude of the slower component results in a steady-state inactivation curve shifted to more negative potentials and similar to the curve produced by Kv1-C (Fig. 5B and D). Using the amplitude of the fast component results in a curve shifted further to the left with a steeper slope (Fig. 5D).

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Figure 5. Kv C- and N-termini produce distinct changes in Kv1.4 steady-state inactivation A, B and C show whole cell currents in oocytes held at -90 mV, pulsed to -10 mV in 10 mV steps of 500 ms duration, subsequently pulsed to +70 mV for 200 ms and repolarized to -90 mV for 15 s. D, steady-state inactivation-voltage curves. For Kv1.4 and Kv1.4 + Kv-C, curves were generated from currents at +70 mV, normalized to the maximum and plotted against test potential. For Kv1.4 + Kv1.2, currents were divided into fast and slow components by fitting as a sum of two exponential functions. Values of peak currents were determined for both components at +70 mV, normalized to the maximum values and both sets of data plotted against test potential. Data were fitted to a Boltzmann equation as described in Fig. 2. The following values were obtained for half-maximal activation (V0·5) and k, respectively: -35·05 ± 0·76, -7·35 ± 0·23 mV (Kv1.4, ; n = 8), -42·06 ± 0·97, -6·59 ± 0·08 mV (Kv1.4 + Kv1-C, ; n = 6), -44·31 ± 1·49, -6·79 ± 0·53 mV (Kv1.4 + Kv1.2 slow component, ; n = 6) and -47·72 ± 1·42, -4·60 ± 0·38 mV (Kv1.4 + Kv1.2 fast component, ; n = 6).

Separable effects of Kv1.2 N- and C-termini on deactivation of Kv1.4

Tail currents of Kv1.4 alone or in the presence of Kv1-C and Kv1.2 are shown in Fig. 6A. The tails were fitted with a single exponential function and the time constants plotted against voltage (Fig. 6B). At potentials of -90 mV, deactivation time constants were 4·97 ± 0·08 (n = 6), 5·56 ± 0·04 (n = 6) and 16·82 ± 0·79 ms (n = 5) for Kv1.4 alone, Kv1.4 plus Kv1-C and Kv1.4 plus Kv1.2, respectively. The rate of Kv1.4 deactivation was not affected by Kv1-C whereas Kv1.2 produced a clear slowing of deactivation.

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Figure 6. Kv1.2 slows deactivation of Kv1.4 A, normalized whole cell currents measured in Xenopus oocytes injected with cRNAs for Kv1.4 alone or with Kv1.2 or Kv1-C. Oocytes were held at -80 mV, pulsed to +80 mV for 125 ms and repolarized to -80 mV. B, plot of deactivation time constants versus test potential. Deactivation time constants were calculated by fitting the tail currents with a single exponential function. , and represent time constants of currents produced by injection of Kv1.4 with Kv1.2 (n = 5), Kv1.4 with Kv1-C (n = 6) and Kv1.4 alone (n = 6), respectively.

Effects of Kv1.2 N-terminal ball domain on recovery from inactivation of Kv1.4

Castellino et al (1995) have shown that Kv1.2 slows recovery from inactivation of Kv1.4. To separate C- and N-terminal effects, we compared the effects of Kv1.2, Kv1-C and Kv2 on Kv1.4 recovery from inactivation (Fig. 7). Recovery was slowed equally by Kv1.2 and Kv1-C, indicating that block by the N-terminus ball of Kv1.2 does not contribute to this effect (Fig. 7A). Kv2 also slowed recovery from inactivation of Kv1.4 (Fig. 7B).

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Figure 7. Kv1.2 slows recovery from inactivation of Kv1.4 through its C-termini and not through its N-termini Whole cell currents were measured in Xenopus oocytes injected with cRNA for Kv1.4 alone, Kv1.4 with Kv1-C, Kv1.4 with Kv1.2 and Kv1.4 with Kv2. Oocytes were held at -90 mV, pulsed to +40 mV for 1 s, repolarized to -90 mV for times ranging from 1 to 8 s (interpulse duration) and depolarized again to +40 mV. A, plot of the amount of inactivated current during the second pulse normalized to the amount of inactivated current during the first pulse versus the interpulse duration. Recovery time constants were 1·47 ± 0·07, 3·31 ± 0·25 and 3·32 ± 0·07 s for Kv1.4 alone (; n = 6), Kv1.4 with Kv1.2 (; n = 5) and Kv1.4 with Kv1-C (; n = 5), respectively. B, recovery from inactivation for Kv1.4 alone (; n = 4) and for Kv1.4 with Kv2 (; n = 4). Recovery time constants were 1·18 ± 0·08 and 1·89 ± 0·09 s, respectively.

Yeast two-hybrid interactions

Conserved sequences in the C-terminus of Kv1.2 or Kv2 have been shown to interact with the N-terminus of Kv1 (amino acids (aa) 1-305 or aa 147-305) (Yu et al. 1996; Sewing et al. 1996; Nakahira et al. 1996; Wang et al. 1996). However, there is no evidence regarding the interactions of these Kvs with the variable N-terminal region of Kv1.4 (aa 1-176) that includes the -ball domain. Since changes in inactivation kinetics by the C-terminus of Kv1.2 were manifest (Fig. 4) we tested for a possible interaction between Kv1.2-C and the N-terminus ball domain of Kv1.4 (aa 1-176) using the yeast two-hybrid assay. We found that Kv1-C and the Kv1.4N2-146 domain (aa 1-176) did not interact whereas interactions were present for Kv1-C and the N-terminus of Kv1.4 downstream from the -ball domain (aa 147-305) (Fig. 8).

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Figure 8. Yeast two-hybrid interaction of Kv1.4 N-terminal fragments with Kv subunits -Galactosidase filter lift assay (column labelled X-Gal) of Y190 yeast co-transformed with combinations of DNA binding domain (BD) and activation domain (AD) plasmids. Development of a blue colour within 8 h indicated an interaction between the two fusion proteins (scored as a + or - to the right of the X-Gal column).

	DISCUSSION

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Kv subunit C-terminal domains increase functional expression of the inactivating K⁺ channel, Kv1.4

We have called the Kv1 C-terminus the '-expression domain' because this region is responsible for altering the functional expression of Kv1·2 and Kv1·5 (Accili et al. 1997a). Using the non-inactivating Kv1.4 mutant with amino acids 2-146 deleted, we showed that Kv1-C, which is the C-terminal 329 amino acids of the Kv1 family, produced a significant increase in the currents expressed by this channel. Taken together with the evidence from yeast two-hybrid assays (Yu et al. 1996; Wang et al. 1996) and other binding studies (Sewing et al. 1996) the functional effects of Kv1-C or Kv2 on expression probably result from a direct interaction with the N-terminus of Kv1.4.

The increase in Kv1.2 produced by Kv is the result of an increase in the number of functional channels in the membrane (Accili et al. 1997a). Single channel measurements of non-inactivating Kv1.4 mutants similar to the mutant used here have shown that the mean open probability is approximately 80 % at the test potentials we have used (Tseng-Crank et al. 1993). Assuming no change in single channel conductance, the increase in Kv1.4 current produced by Kv-C in this study was also probably due to an increase in the number of functional channels. That Kv1.4 single channel conductance was unchanged is reasonable since no changes in Kv1.2 single channel conductance were observed upon co-expression with Kv1-C (Accili et al. 1997a). The increase in Kv1.4 may be due to a more efficient maturation of the subunits leading to an increase in the surface expression as suggested for non-inactivating Kv1 subunits (Shi et al. 1996).

Separable effects of Kv1.2 N- and C-termini on inactivation of Kv1.4

Our results show that co-expression of Kv1-C with Kv1.4 increased the rate of inactivation and slowed the rate of recovery from inactivation. The latter effect differs from that observed for Kv2 and rat Kv1.4 in the presence of Rb⁺ (McIntosh et al. 1997). Our results agree with previous studies which have shown that recovery from inactivation was slowed by Kv1.2 only for Kv1.4 and not for Kv1.4N2-146 (Castellino et al. 1995). In the absence of Kv1.2, the rate of recovery from inactivation was not different between Kv1.4 and Kv1.4N2-146 (Rasmusson et al. 1995). These authors concluded that the slow recovery was dominated by C-type inactivation and that the Kv1.4 N-terminus was required for the increased inactivation and slowed recovery.

A possible explanation for such results is that Kv1-C, i.e. the C-terminus of Kv, interacts with the N-terminal -ball domain of Kv1.4. However, our yeast two-hybrid assay results do not support this explanation. Since the Kv C-terminus does bind to the Kv1.4 N-terminus downstream from the -ball we propose that Kv1-C or Kv2 produces an allosteric enhancement of -ball blockade.

The inactivation introduced by the -ball on non-inactivating Kv1 channels has been attributed to block of open channels (DeBiasi et al. 1997; Accili et al. 1997a). The results we obtained using the non-inactivating mutant of Kv1.4 probably have the same basis. As for the naturally non-inactivating Kv1 channels, Kv1-C and Kv2 also had no effects on voltage-dependent activation or deactivation.

Block by the -ball was observed for intact Kv1.4 where an additional voltage-dependent component of inactivation significantly reduced peak outward current. A hyperpolarizing shift and increase in slope of the steady-state inactivation curve were also observed. These effects may also be explained by open channel block. The effects of Kv1.2 on deactivation may be due to unblock of the N-terminus -ball. Previous experiments using Kv1.4 and an N-terminal deletion mutant of Kv1.4 have shown that the presence of the -ball has little effect on deactivation (Bertoli, Moran & Conti, 1994).

A slowing of block produced by the -ball was observed in the presence of the -ball and vice versa. An apparent slowing would be expected if block by each ball was mutually exclusive. In fully activated channels the rate of block by the -ball was voltage dependent whereas that of the -ball was not, suggesting differences in binding site or access between the two balls.

An increase in the rate of C-type inactivation by Kv1.2 was observed at 5 mM external K⁺ concentration, as described previously (Morales et al. 1996). Kv1-C and Kv2 had no effect on C-type inactivation, implicating block by the -ball in this effect. However, we found that at 50 mM K⁺, C-type inactivation was unaffected by Kv1.2. Thus, the open channel block by the -ball probably inhibits K⁺ efflux as suggested for the -ball effect on C-type inactivation of Shaker K⁺ channels (Baukrowitz & Yellen, 1995).

A model of Kv effects on Kv1.4

Figure 9 was adapted from Hoshi et al. (1991) and shows how we envision inactivation in a hetero-oligomeric Kv1.4-Kv1.2 channel. The model can also explain the effects produced by a mixture of inactivating and non-inactivating Kv subunits (Accili et al. 1997b; Xu & Li, 1997). The channel goes from closed to open (state A to state B) upon depolarization and then can either be blocked by the -ball (state C), or undergo C-type inactivation (state E). Since block by the -ball enhances C-type inactivation (Hoshi et al. 1991; Baukrowitz & Yellen, 1995; Rasmussen et al. 1995) the channel assumes state D. Kv1.2, through its C- and N-termini, produces two separate effects on inactivation. Kv1-C or Kv2 enhances the block produced by the allosterically modified -ball. Activation and deactivation kinetics between A and B are not affected, but the B to C transition rate for the -ball is increased. The recovery from inactivation is thought to be rate limited by recovery from C-type inactivation (Rasmussen et al. 1995). In our model, increased allosterically enhanced block by the -ball increases the transition rate constants for E to D and/or C to D. Block by the -ball is produced by the transition from B to state F. The channel may be blocked by either ball but not by both. It is this interference that slows the blocking rate of either ball. State F is not coupled to E or D since no effects of -ball blockade on C-type inactivation were observed in our experiments.

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Figure 9. Proposed scheme for Kv1.2 effect on the inactivation of Kv1.4 The scheme shows a cutaway of an hetero-oligomeric Kv1.4-Kv1.2 channel in the following states: closed (A), open (B), blocked by the Kv1.2 N-terminus -ball domain (F), blocked by the Kv1.4 N-terminus -ball domain (C), undergoing C-type inactivation (E) and undergoing C-type inactivation during block by the Kv1.4 N-terminus -ball (D). Kv1.2 is bound to the region of the Kv1.4 N-terminus defined by yeast two-hybrid and co-immunoprecipitation data. For simplicity, the cutaway shows two -subunits, one N-terminus -ball domain and one Kv binding site and Kv N-terminus -ball.

Functional roles of Kv subunits in modifying inactivating K⁺ channels in neurons

Transient K⁺ currents contribute to the regulation of firing frequency of neurons (Connor & Stevens, 1971) and more recently have been implicated in regulating action potential propagation in axons (Debanne et al. 1997). Heterologous expression of Kv1.4 in Xenopus oocytes alone, or with Kv subunits, produces inactivating currents similar to those found in central neurons, suggesting that this channel makes an important contribution to neuronal A-currents (Pardo et al. 1992; Rettig et al. 1994). Evidence exists for extensive co-localization of Kv1.4 and Kv subunits in cell bodies and axons of central neurons (Rhodes et al. 1995, 1997; Nakahira et al. 1996). Homomultimers of Kv1.4, and heteromultimers of Kv1.4 with Kv subunits and/or other Kv1 subunits have been characterized from central neurons (Shamotienko et al. 1997). Our previous (Accili et al. 1997b) and present results, and the results of others (Castellino et al. 1995), offer several potential roles for Kv subunits in modifying the electrical properties of central neurons by acting on inactivating K⁺ channels. Kv subunits can increase the functional expression of inactivating K⁺ channels. Non-inactivating Kv subunits can increase inactivation rates and slow recovery from inactivation of inactivating K⁺ channels. Inactivating Kv subunits also slow recovery, through their C-termini, as well as producing additional open channel block reducing peak current and slow deactivation. These mechanisms greatly expand the mode and range over which inactivating K⁺ channels, and hence neuronal firing frequency and action potential propagation, can be regulated and underline the importance of analysing the functional relationship between inactivating K⁺ channels and Kv subunits in vivo.

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Acknowledgements

We thank Drs A. E. Lacerda and E. Ficker for discussion, and Mr Tom Carroll and Dr W. Q. Dong for technical assistance. This work was supported by NIH grants HL-37044, HL-36930 and NS-23877 (A. M. B.) and a grant from the American Heart Association, Northeast Ohio Affiliate (B. A. W.).

Corresponding author

Y. A. Kuryshev: Rammelkamp Center, 2500 MetroHealth Drive, Cleveland, OH 44109-1998, USA.

Email: ykuryshev{at}research.mhmc.org

Author's present address

E. A. Accili: School of Kinesiology, Simon Fraser University, Vancouver, British Columbia, Canada V5A 1S6.

E. A. Accili and Y. A. Kuryshev contributed equally to this work.

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