©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Subunit Increases Rate of Inactivation of Specific Voltage-gated Potassium Channel Subunits (*)

(Received for publication, November 1, 1994; and in revised form, January 23, 1995)

Michael J. Morales (1) Robert C. Castellino (2) Anne L. Crews (3) Randall L. Rasmusson (4) Harold C. Strauss (1) (3)(§)

From the  (1)Department of Pharmacology, (2)Department of Cell Biology, and (3)Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710 and the (4)Department of Biomedical Engineering, School of Engineering, Duke University, Durham, North Carolina 27708

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Voltage-gated potassium channel beta subunits are cytoplasmic proteins that co-purify with the pore-forming alpha subunits. One of these subunits, Kvbeta1 from rat brain, was previously demonstrated to increase the rate of inactivation of Kv1.1 and Kv1.4 when co-expressed in Xenopus oocytes. We have cloned and characterized a novel voltage-gated K channel beta subunit. The cDNA, designated Kvbeta3, has a 408-amino acid open reading frame. It possesses a unique 79-amino acid N-terminal leader, but is identical with rat Kvbeta1 over the 329 C-terminal amino acids. The Kvbeta3 transcript was found in many tissues, but was most abundant in aorta and left ventricle of the heart. Co-expression of Kvbeta3 with K channel alpha subunits shows that this beta subunit can increase the rate of inactivation from 4- to 7-fold in a Kv1.4 or Shaker B channel. Kvbeta3 had no effect on Kv1.1, unlike Kvbeta1 which can increase rate of inactivation of this alpha subunit more than 100-fold. Other kinetic parameters were unaffected. This study shows that voltage-gated K channel beta subunits are present outside the central nervous system, and that at least one member of this family selectively modulates inactivation of K channel alpha subunits.


INTRODUCTION

Voltage-gated potassium channels are present in all excitable and most nonexcitable eukaryotic cell types. They are involved in a diverse array of functions, including electrogenesis, secretion, and cell motility. In mammals, a diverse group of more than 50 members of an extended gene family homologous to the Shaker gene of Drosophila have been identified that are responsible for generating many voltage-gated K currents(1, 2, 3, 4, 5) .

Shaker-type K channel proteins, expressed in Xenopus oocytes or mammalian cell lines, form tetramers of alpha subunits that selectively conduct K ions and reproduce many properties of native channels. However, there are inconsistencies between native and cloned K channel currents, suggesting a failure to reconstitute the native channels fully in heterologous expression systems. These inconsistencies have several possible explanations. Some native K channels are heteromultimeric associations of more than one type of alpha subunit. These associations can modify the current phenotypes, making them distinct from currents produced by either alpha subunit alone(6, 7, 8) . It has also been suggested that differences between cloned and native channels are due to differences in post-translational processing or intracellular environment between heterologous expression systems and the channel's native environment(9, 10, 11) . Finally, K channel structure in heterologous expression systems may be incomplete(12, 13) . Two voltage-gated K channels recently purified from mammalian brain (14, 15) were shown to consist of an alpha subunit, identical with previously described Shaker-like K channel proteins, and a smaller beta subunit (M(r) = 40,000). This suggests that complete reconstitution of K channels in heterologous expression systems requires the presence of appropriate beta subunits.

Partial amino acid sequencing of the bovine K channel beta subunit led to the cloning of one bovine brain (14) and two rat brain (16) beta subunit cDNAs. They were divided into two classes based on differences in sequence: Kvbeta1, found in rat, and Kvbeta2, found in both rat and bovine. These beta subunits consisted of 401 and 367 amino acids, respectively, and had 85% identity in their 329 C-terminal amino acids. The respective N termini of 72 and 38 amino acids exhibit no conservation. Co-expression of rat Kvbeta1 with a delayed rectifier type K channel (RCK1; Kv1.1) increased the rate of inactivation more than 100-fold. Site-directed mutagenesis showed that the increase in inactivation rate was mediated through the N-terminal region of the beta subunit. The Kvbeta2 subunit did not affect the properties of RCK1 or of a fast-inactivating Kv1.4 channel, RCK4(16) .

Kvbeta1 mRNA was detected exclusively in brain(16) . To determine if novel K channel beta subunits might be found in other tissues, we attempted to clone these proteins from heart. The large array of K currents in heart (17, 18) made it a logical source of unknown beta subunits. A cDNA was isolated from a ferret ventricular cDNA library. It contained a single open reading frame highly conserved with previously described voltage-gated K channel beta subunits(16, 19) . Its deduced amino acid sequence contained a unique leader sequence, followed by 329 amino acids identical with rat Kvbeta1. The Kvbeta3 transcript was most abundant in aorta and left ventricle, although it was also found in other locations in the heart as well as brain, skeletal muscle, and kidney. Kvbeta3 is conserved in humans and rats. Kvbeta3 was co-expressed in Xenopus oocytes with K channel alpha subunits from ferret (Kv1.4(20) ), rat (Kv1.1(21) ), and Drosophila (Shaker BDelta6-46(22) ). It accelerated inactivation in Kv1.4 in a manner analogous to that of Kvbeta1(16) . However, unlike Kvbeta1, which increased the inactivation rate of Kv1.1 approx100-fold, Kvbeta3 had no effect on this alpha subunit. As Kvbeta3 was also capable of modulating inactivation of Shaker B, the failure of Kvbeta3 to alter inactivation of Kv1.1 was not due to strict specificity for Kv1.4 alpha subunits.


MATERIALS AND METHODS

Unless otherwise specified, standard molecular biological methods were used(23, 24) . Enzymes were used according to manufacturers' directions.

Cloning of Kvbeta3

Ferret heart RNA was isolated as described(25) ; 5 µg were reverse-transcribed using Superscript reverse transcriptase (Life Technologies, Inc.) with oligo(dT) primers as described by the manufacturer. Oligonucleotides were designed based on the amino acid sequences NIIKKKGW and NQGMAMYW from amino acids 101-108 and 178-185, respectively, of the Kvbeta2 subunit from bovine brain ((19); Fig. 1). The oligonucleotide sequences were: 5`-TCGAATTCAAYCAGGGMATGGCIATGTAYTGG-3` and 5`-GACCTCGAGCCYTCGTTICKIARRCACCA-3` (R = A/G; M = A/C; I = inosine; K = G/T; Y = C/T). PCR (^1)was performed as described(26) , with 1/20 of the cDNA from the reverse transcription reaction. The cycling parameters utilized ``Touchdown PCR''(27) , with a starting annealing temperature of 55 °C, and a concluding annealing temperature of 42 °C for the final 10 cycles. A probe was made by random-priming the PCR product with digoxigenin-11-dUTP to allow chemiluminescent detection (Boehringer Mannheim). The random-primed PCR product was used to probe a ferret ventricle cDNA library (20) constructed in ZAPII (Stratagene). Plaque lifts, hybridization, and detection were performed as described by Boehringer Mannheim. The positive plaque, FHBK1, was rescued by in vivo excision; the resulting plasmid, pFHBK1, contained the full length of the beta subunit open reading frame and about 1.5 kilobase pairs of 3`-untranslated region, including the poly(A) tail. Single-stranded phagemid DNA (28) was utilized for sequencing with Sequenase 2.0 (U. S. Biochemicals) using custom oligonucleotides. Each nucleotide on both strands was unambiguously identified at least once. Sequence similarity searches were performed through the NCBI BLAST server(29) .


Figure 1: Deduced amino acid sequence of ferret Kvbeta3. The sequence is shown aligned with the other known K channel beta subunit sequences: rat Kvbeta1, rat Kvbeta2, and bovine Kvbeta2(16, 19) . Amino acids identical with those in Kvbeta3 are designated with a hyphen in the Kvbeta1 and -2 sequences. The nucleotide sequence of ferret Kvbeta3 has been deposited in GenBank under the accession number U17966.



Human and rat Kvbeta3 cDNAs were isolated by standard PCR of cDNA synthesized as above. Human heart whole cell RNA was purchased from Clontech. The primers used were FKbeta3/58 (5`-GTATAAACCTGCCTGTGC-3`; covering amino acids 4-9 in ferret Kvbeta3) and FKbeta3/37 (5`-AGCCTTTCCAGCAGCATAGACTTC-3`; covering amino acids 129-135; Fig. 1). PCR was performed as described(30) . After a 5-min 94 °C denaturation step, the reactions were cycled 35 times at 94 °C for 30 s, 53 °C for 45 s, and 72 °C for 1 min. The procedure was completed with a 5-min incubation at 72 °C. The PCR products were cloned into pBS-SK, and two independent clones of each were sequenced. Partial cDNA sequences of the rat and human Kvbeta3 have been deposited in GenBank under the accession numbers U17967 and U17968, respectively.

Competitive PCR

Whole cell RNA was prepared from ferret heart, brain, liver, kidney, skeletal muscle(25) , aorta, and aortic endothelial cells(31) . Endothelial cells were isolated and cultured as described(20) . Complementary DNA was made from 5 µg of RNA using SuperScript reverse transcriptase with a random hexamer primer (Life Technologies, Inc.). Competitive PCR was performed essentially as described(32) . One-twentieth of each reverse transcriptase reaction (along with a sham cDNA control lacking reverse transcriptase) was amplified by PCR. Each reaction contained 2 µM primer FKbeta3/37, 2 µM primer FKbeta3/56 (5`-TCTCAGAGCTAAAGACTGTGAAATGAGC-3`; identical with nucleotides 4-31 upstream of the translational start site), and 0.2 fmol internal standard. The cycling parameters were the same as for isolation of rat and human Kvbeta3, except the annealing temperature was 58 °C. The reaction products were separated on a 5% polyacrylamide, 1 times TBE gel, stained with ethidium bromide, and photographed under ultraviolet light. The 272-bp internal standard was synthesized as described (33) using primers FKbeta3/56 and FKbeta3/37 (5`-AGCCTTTCCAGCAGCATAGACTTCATTCCTGTAAGCCATG-3`; the first 24 nucleotides are identical with FKbeta3/37, the last 16 nucleotides are complementary to nucleotides 286-301; see Fig. 2A).


Figure 2: A, strategy for detection of Kvbeta3 transcripts by competitive PCR. Ferret transcripts were detected using oligonucleotides Kvbeta3/56 and Kvbeta3/37, which give a 460-bp DNA molecule (B). The internal standard, added to PCR reactions in B, was a 183-bp deletion of Kvbeta3 that preserved the nucleotide sequence complementary to the oligonucleotides; its product was 272 bp. The open boxes represent unconserved regions, and the shaded boxes represent the conserved region of Kvbeta3. B, competitive PCR of cDNAs from various ferret tissues. Five µg of whole cell RNA were reverse-transcribed and amplified by PCR in the presence of 0.2 fmol internal standard. Lanes 1, cDNA alone; 2, internal standard alone; 3, right ventricle; 4, left ventricle; 5, atrium; 6, aorta; 7, cultured endothelial cells; 8, brain; 9, skeletal muscle; 10, liver; and 11, kidney. Competitive PCR of sham reverse transcription controls showed no detectable signal (data not shown).



K Current Measurement

Xenopus laevis oocytes (stage V-VI) were defolliculated by gently shaking for 3 h in Ca-free OR2 solution (in mM: 82.5 NaCl, 2 KCl, 1 MgCl(2), 5 HEPES-NaOH, pH 7.40) with 1-2 mg/ml collagenase(20) . Oocytes were injected with 50 nl of cRNA solution containing up to 50 ng of RNA with and without beta subunit at ratios varying from 1:1 (alpha:beta) to 1:16, as described previously (20) . Voltage-clamp experiments were conducted on oocytes within 3-6 days of injection using a two electrode ``bath clamp'' amplifier (OC-725A, Warner Instruments) as described previously. Briefly, bath potential was sensed using an agar bridge electrode, and bath potential was maintained using a separate current passing agar bridge electrode. The amplifier was used with the ``DC gain feature'' which reduced errors due to finite open loop gain. Average peak current size at +50 mV for all analyzed currents was 7.4 ± 0.9 µA (n = 118). No correlation between current size and inactivation rate was detected for the range of currents measured in this study. Extracellular solution was ND-96 (in mM: 96 NaCl, 2 KCl, 1 MgCl(2), 1.8 CaCl(2), 5 HEPES-NaOH, pH 7.4). Electrodes were filled with 3 M KCl (resistances 0.6-1.5 megaohms) and shielded to reduce capacitive coupling. All measurements were carried out at room temperature. Data were not leakage- or capacitance-subtracted, unless otherwise specified. Activation was measured using the cut-open oocyte technique(34) , as described previously(20) . Data from two-electrode voltage clamp were filtered at 2 kHz or 5 kHz for cut-open oocyte and analyzed using an Axon Instruments TL-1 interface and pClamp software. Confidence levels were calculated using an unpaired t test.


RESULTS AND DISCUSSION

Fig. 1shows an alignment of the deduced amino acid sequence of ferret Kvbeta3 to K channel beta subunits from rat (Kvbeta1 and Kvbeta2) and bovine (Kvbeta2). Strikingly, Kvbeta3 and rat Kvbeta1 are identical over the last 329 amino acids, a region where Kvbeta1 and Kvbeta2 share 85% identity. However, there is no similarity among the first 79 amino acids of the ferret beta subunit and the first 72 or 38 amino acids of Kvbeta1 and Kvbeta2, respectively. Similarity searches of both the entire beta subunit and the N-terminal 79 amino acids revealed no significant similarity between ferret Kvbeta3 and any other protein except the previously described beta subunits. No similarity was found between the N terminus of ferret Kvbeta3 and any other protein sequence. Despite their apparent lack of similarity, the N termini of Kvbeta1 and Kvbeta3 share one potentially important structural feature: a cysteine residue near the N terminus. Oxidation of this amino acid, also found near the N termini of other fast inactivating channels, can dramatically decrease inactivation rates (11, 16) . Hydropathy and secondary structural analyses of the Kvbeta1 and Kvbeta3 N termini fail to show any apparent similarity in predicted secondary structure(35, 36, 37, 38, 39, 40) . Hydropathy analysis also suggested Kvbeta3 is a cytoplasmic protein, as has been predicted for Kvbeta1 and Kvbeta2 (16, 19) . Comparison of the deduced Kvbeta3 amino acid sequence with protein motif data bases (41, 42) showed the presence of 33 consensus phosphorylation sites.

To determine the tissue localization of Kvbeta3, competitive PCR (32) was performed on cDNA made from several tissues. Oligonucleotides were chosen at the 5` end of the clone to ensure specific amplification of Kvbeta3 (Fig. 2A). Ferret Kvbeta3 transcript was most abundant in aorta followed by left ventricle (Fig. 2B). Transcripts were also detected in right ventricle, atrium, brain, skeletal muscle, and kidney. None were detectable in liver or cultured aortic endothelial cells. Aorta is rich in smooth muscle, fibrous tissue, and endothelium; as the transcript was not present in endothelial cells, it is likely that this transcript is present in smooth muscle or fibroblasts, although its presence in other cell types cannot be ruled out.

Kvbeta3 is not unique to ferret. Cross-species PCR using oligonucleotides FKbeta3/37 and FKbeta3/58 (which is complementary to the unique portion of the ferret cDNA) was performed on rat and human heart cDNA. The reactions yielded the expected 400-nucleotide DNA species, which was cloned and sequenced. There was extensive similarity in the nucleotide sequence among ferret, human, and rat, showing that Kvbeta3 is present in all three species. The amino acid sequence conservation in the N-terminal region was 88% between ferret and human and 80% between ferret and rat (data not shown).

Kvbeta1 can increase the rate of inactivation of both a delayed rectifier and a fast-inactivating (A-type) K channel alpha subunit(16) . Conservation between Kvbeta3 and Kvbeta1 suggested that Kvbeta3 might have a similar influence. To test the effect of Kvbeta3 on inactivation and other channel properties, it was co-expressed in Xenopus oocytes with FK1, a ferret Kv1.4 channel(20) , and RCK1 (rat Kv1.1(21) ). Co-injection of Kvbeta3 mRNA with FK1 mRNA caused subtle changes in inactivation kinetics (Fig. 3A). Inactivation of FK1 channels has been previously shown to be biexponential, with two closely spaced time constants at +50 mV(20) . The time constant of the fast component of inactivation decreased from 42 ± 3 (n = 20) for FK1 alone to 6.2 ± 0.2 ms (n = 28); the slow component decreased from 313 ± 94 to 101 ± 2 ms. The decrease of the fast component of inactivation was comparable to the overall decrease induced by Kvbeta1 expressed with rat Kv1.4. Co-expression of Kvbeta3 with FK1 also changed the relative contribution of each component of inactivation; the ratio of the amplitude of fast to slow time constants decreased from 5.0 ± 1.7 to 0.67 ± 0.03. Because of the greater participation of the slow component, the net result was a minor alteration in the overall rate of inactivation.


Figure 3: Outward K currents elicited by depolarizing pulses to +50 mV from a holding potential of -90 mV using a two-electrode voltage clamp. Currents were normalized relative to the same peak current to emphasize the kinetic changes induced by co-expression of Kvbeta3. Currents resulted from depolarizations to +50 mV from a holding potential of -90 mV, stimulation rate of 0.1 Hz, and perfused with ND-96. Oocytes were injected with mRNA encoding various K channel subunits types. A, FK1 alone (Kv1.4, 8 ng of mRNA/oocyte, peak current 12.7 µA) or FK1 + Kvbeta3 (8 and 32 ng of mRNA/oocyte, peak current 9.7 µA). Note that expression of FK1 alone results in a smooth inactivation with a small but slow late component of inactivation. Co-expression of FK1 with Kvbeta3 results in an initial small rapid component followed by a much larger slow component of inactivation. B, RCK1 alone (Kv1.1, 2 ng of mRNA/oocyte, peak current 14.4 µA) or RCK1 + Kvbeta3 (2 and 32 ng of mRNA/oocyte, respectively, peak current 10.2 µA). Co-injection of Kvbeta3 with RCK1 did not increase the rate of inactivation. C, FK1Delta2-146 (8 ng of mRNA/oocyte, peak current 5.7 µA), an N-terminal deletion mutant (see (20) ) or FK1Delta2-146 + Kvbeta3 (2 and 32 ng of mRNA/oocyte, respectively, peak current 1.7 µA). Co-injection of FK1Delta2-146 with Kvbeta3 partially, but not completely, restored inactivation. D, Shaker BDelta6-46 alone (2 ng of mRNA/oocyte, 9.1 µA) or Shaker BDelta6-46 + Kvbeta3 (8 and 32 ng of mRNA/oocyte, respectively, peak current 11.1 µA). The increase in inactivation rate was similar to the results with FK1Delta2-146. Kvbeta3 partially restored inactivation in the mutant Shaker channel and induced a very small additional fast component that was not observed with FK1Delta2-146.



In contrast to the results obtained with FK1, co-injection of RCK1 (Kv1.1) with Kvbeta3 failed to show any effect on channel inactivation (Fig. 3B). This was true despite a 16:1 (by weight) co-injection of Kvbeta3:RCK1 mRNA; parallel experiments using FK1 and FK1Delta2-146 (see below) showed that the Kvbeta3 subunit was active (data not shown). These results are strikingly different from those obtained by Rettig et al.(16) with Kvbeta1. They showed an approximately 100-fold increase in the rate of inactivation of RCK1. Since Kvbeta3 was co-injected with the identical RCK1 alpha subunit, and conservation of RCK4 and FK1 is 97%(20) , it is likely that the difference in inactivation is due to the different N termini of Kvbeta3 and Kvbeta1.

It is possible that the change in rate of inactivation caused by Kvbeta3 may be dependent on the ratio of expressed alpha subunit to beta subunit. If the beta subunits failed to saturate all available binding sites on the alpha subunits, the overall rate and degree of beta subunit-mediated inactivation would likely be decreased. A second explanation is that the maximal association of beta subunits to alpha subunits was achieved, but that Kvbeta3 simply induced slower inactivation, or perhaps caused inactivation by a different mechanism. To discriminate between these possibilities, a mutant form of FK1, FK1Delta2-146, in which the first 146 N-terminal amino acids have been deleted to remove fast N-type inactivation ((20); referred to as FK1DeltaNco), was co-expressed with Kvbeta3. As shown in Fig. 3C, co-expression with Kvbeta3 greatly increased the rate at which FK1Delta2-146 inactivated. The time constant of inactivation for FK1Delta2-146 was 1795 ± 81 ms (at +50 mV), decreasing to 409 ± 27 ms with co-injection of Kvbeta3 mRNA. The effects of co-expression of Kvbeta3 mRNA on the inactivation rate of FK1Delta2-146 were dependent upon the ratio of injected Kvbeta3 to FK1Delta2-146 mRNA for ratios less than 2 to 1 but were insensitive to ratios higher than 2 to 1 (Fig. 4), suggesting that the beta subunits were saturating at the mRNA concentrations used in these experiments.


Figure 4: Saturation of FK1Delta2-146 with Kvbeta3. The alteration of inactivation time constants was sensitive to the ratio of beta subunit to alpha subunit for low ratios but saturated for high ratios of beta to alpha subunit, indicating that the rate of inactivation reflected the maximal association of Kvbeta3 to FK1Delta2-146 channels. Two ng/oocyte of FK1Delta2-146 was injected into each oocyte followed by a second injection of 0, 2, 4, 8, 16, or 32 ng/oocyte Kvbeta3 mRNA. Inactivation was measured with a single exponential time constant fit to the rate of current decay. The data for each particular level of injected beta subunit mRNA is given as the mean ± S.E. (n = 11, 4, 8, 11, 6, 10 oocytes for each point, average peak currents were 4.9 ± 0.5, 3.7 ± 0.8, 3.1 ± 0.4, 2.3 ± 0.2, 2.4 ± 0.2, 1.5 ± 0.1 µA, respectively).



These data showed that ferret Kvbeta3 can associate with ferret Kv1.4 (FK1), but did not rule out the possibility that this beta subunit's effects might be specific for only a limited class of alpha subunits. Therefore, we co-injected Kvbeta3 with the distantly related channel, Shaker B from Drosophila. Since this channel is only 50% conserved with FK1, it appeared to be an unlikely candidate to associate with a mammalian beta subunit. We used Shaker BDelta6-46(22) , a mutant lacking fast N-type inactivation, to be able to clearly detect the effects of beta subunit association. Co-expression of Shaker BDelta6-46 with Kvbeta3 resulted in an increase in the degree and rate of inactivation similar to that observed for FK1Delta2-146 (Fig. 3D). Association with Shaker B suggested that Kvbeta3 is probably binding to the much more highly conserved RCK1, but for some reason is unable to induce inactivation.

Other gating characteristics of FK1 and FK1Delta2-146 were not strongly altered by co-expression of Kvbeta3. Fig. 5shows data obtained using the cut-open oocyte clamp (34) for FK1Delta2-146 in the presence and absence of beta subunit. FK1Delta2-146 was used to minimize the overlap of fast inactivation with activation. There was no obvious difference in the sigmoid onset of the current during the first 2 ms at +50 mV; however, the late rising phase of current was terminated prematurely by what appears to be the onset of an inactivation-like process. Although the time to peak current appears to have decreased with co-expression of Kvbeta3, this is most likely due to the increased inactivation rate. Similarly, beta subunit expression had only modest effects on recovery from inactivation when co-expressed either with full-length FK1 (t = 4.88 ± 0.13 s (n = 4) with beta subunit; t = 2.95 ± 0.18 s (n = 4) without beta subunit) or FK1Delta2-146 (t = 2.94 ± 0.12 s (n = 5) with beta subunit; t = 2.20 ± 0.11 s (n = 4) without beta subunit).


Figure 5: Early onset of current activation is unchanged by the addition of beta subunit. Currents recorded from FK1Delta2-146 (solid line) and FK1Delta2-146 co-expressed with Kvbeta3 (dashed line) using the cut-open oocyte voltage clamp technique as described(20, 34) . The currents were normalized for the purpose of comparison. The peak current occurred much earlier due to the overlap with beta subunit-mediated inactivation, but was otherwise apparently unaffected. Current amplitudes were 15 and 2.1 µA for FK1Delta2-146 and FK1Delta2-146 + beta subunit, respectively. Both currents were recorded in response to a depolarizing pulse to +50 mV from a holding potential of -90 mV and were capacitance and leakage compensated. No off-line subtraction procedures were employed.



We have cloned, sequenced, and characterized a novel K channel beta subunit, Kvbeta3. The deduced protein sequence is identical with Kvbeta1 in its 329 C-terminal amino acids, but has a 79-amino acid N terminus with no sequence identity with Kvbeta1 or Kvbeta2. Conservation of C termini between Kvbeta1 and Kvbeta3, and their sharp transition to a nonconserved region, suggested that one mechanism for generating different beta subunits might be alternative splicing of precursor transcripts.

The general structural similarity between Kvbeta1 and Kvbeta3 implied that Kvbeta3 might induce inactivation in K channel alpha subunits. This was true for mutant channels that have had N-type inactivation disrupted (FK1Delta2-146 and Shaker BDelta6-46) and in wild-type Kv1.4. However, unlike Kvbeta1, it did not cause inactivation of a Kv1.1 alpha subunit. The failure of Kvbeta3 to modify RCK1 inactivation kinetics may reflect a failure of this alpha and beta subunit to associate, although association with Shaker argues against this. Alternatively, Kvbeta3 and RCK1 may associate, but specific domains required for inactivation cannot interact.

Acceleration of inactivation by Kvbeta1 is mediated by a ``tethered ball''(16) . If Kvbeta3 operates through a similar mechanism, the implication is that the inactivation domain of Kvbeta3 is specific for certain alpha subunits. This seems unlikely, given the recent demonstration that a variety of inactivation domains can induce inactivation in fast inactivating and non-inactivating alpha subunits (43, 44) . Another possibility is that Kvbeta3 alters inactivation through another mechanism. In either case, it seems clear that the nonconserved N-terminal region in beta subunits confer some degree of specificity of interaction.

Kvbeta3 is the third member of a family of ancillary subunits that associate with voltage-gated K channels. Co-expression of Kvbeta3 causes alterations in K channel function that are distinct from the effects Kvbeta1 or Kvbeta2. This suggests that the diversity of in vivo K channel function may result from expression of different K channel beta subunits, as well as diversity of expression and assembly of alpha subunits. This mechanism is similar to that of Na and Ca channels, which are known to generate diversity through assembly of different subunits(45) . Given the wide variety of associated subunits for these other channels, it seems likely that Kvbeta1, Kvbeta2, and Kvbeta3 are the first members of a large group of ancillary K channel subunits that differ in their N termini. The novel differences in specificity of channel interaction and electrophysiological properties of Kvbeta3 from Kvbeta1 and Kvbeta2 provide an additional basis for understanding K channel function and diversity.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants HL19216 and HL52874. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Jan Tytgat for his gift of the RCK1 clone and Ligia Toro de Stefani for her gift of the Shaker BDelta6-46 clone; Mary Comer and Donald Campbell for discussions and critical comments regarding this manuscript; and David Lamson and Ying Zhang for excellent technical assistance.


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