©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Selective Interaction of Voltage-gated K Channel -Subunits with -Subunits (*)

(Received for publication, November 13, 1995; and in revised form, January 8, 1996)

Kensuke Nakahira Gongyi Shi Kenneth J. Rhodes (1) James S. Trimmer (§)

From the Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215 Central Nervous System Division, Wyeth-Ayerst Research, Pearl River, New York 10965

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To begin to study the molecular bases that determine the selective interaction of the beta-subunits of voltage-gated K channels with alpha-subunits observed in situ, we have expressed these polypeptides in transfected mammalian cells. Analysis of the specificity of alpha/beta-subunit interaction indicates that both the Kvbeta1 and Kvbeta2 beta-subunits display robust and selective interaction with the five members of the Shaker-related (Kv1) alpha-subunit subfamily tested. The interaction of these beta-subunits with Kv1 alpha-subunits does not require the beta-subunit N-terminal domains. Thus, the previously observed failure of N-terminal mutants of Kvbeta1 to modulate inactivation kinetics of Kv1 family members is not simply due to a lack of subunit interaction. Interaction of these beta-subunits with members of two other subfamilies (Shab- and Shaw-related) could not be detected. Somewhat surprisingly, a member of the Shal-related subfamily was found to interact with beta-subunits; however, this interaction had biochemical characteristics distinct from the beta-subunit interaction with Kv1 family members. In all cases, Kvbeta1 and Kvbeta2 exhibited indistinguishable alpha-subunit selectivity. These studies point to a selective interaction between K channel alpha- and beta-subunits mediated through conserved domains in the respective subunits.


INTRODUCTION

Voltage-dependent K channels are fundamental and diverse components of neuronal activity. Molecular cloning studies have identified over a dozen distinct K channel genes and shown that the encoded pore-forming alpha-subunits are members of a large, multigene superfamily that includes Na and Ca channel alpha-subunits(1) . Although expression of these individual alpha-subunits alone is sufficient to generate voltage-gated channels exhibiting many features of the corresponding channels in situ, studies on native Na and Ca channels in neurons and other excitable cells have confirmed the existence of auxiliary polypeptides in tight association with alpha-subunits(2) . Cloning of these auxiliary subunits and their subsequent co-expression with alpha-subunits has shown that the expression level, gating, and conductance properties of expressed channels are profoundly influenced by the presence of auxiliary subunits(2) .

Recently, it has been discovered that K channels also have auxiliary (beta) subunits. A cDNA encoding a beta-subunit copurifying with the bovine brain DTX acceptor complex was recently isolated(3) . Subsequently, cDNAs encoding three highly related yet distinct beta-subunit isoforms were isolated from rat brain (Kvbeta1 and Kvbeta2, (4) ) and from ferret (Kvbeta3, (5) ) and human (hKvbeta3, Refs. 6 and 7) heart. Although dissimilar in their primary structures, beta-subunits of K and Ca channels exhibit general structural similarity in that they are basic (pI approx 9.5), hydrophilic, and presumably peripheral membrane proteins present at the cytoplasmic face of the plasma membrane (2) .

Co-expression of Kvbeta1 was found to greatly accelerate the rate of inactivation of K currents expressed from the Kv1.1 or Kv1.4 alpha-subunit cDNAs in Xenopus oocytes(4) . Kvbeta3, which is an alternatively spliced product of the Kvbeta1 gene, accelerates the rate of inactivation of K currents expressed from Kv1.4 or Kv1.5 but not from Kv1.1, Kv1.2, or Kv2.1 cDNAs(5, 6, 7) . These results suggest that beta-subunit modulation of alpha-subunit gating can contribute additional functional diversity to K channels in excitable cells. Surprisingly, co-expression of the highly related Kvbeta2 had no effect on inactivation, apparently due to the lack of the N-terminal ``ball'' domain present in Kvbeta1 that is both necessary and sufficient for the observed modulation of inactivation(4) . However, from the published electrophysiological analysis of alpha/beta-subunit interaction presented, it was also possible that the lack of observed Kvbeta2 effects was simply due to a lack of Kvbeta2 interaction with the co-expressed alpha-subunits.

We previously used an antibody raised against the C terminus of the bovine beta-subunit, predicted to recognize both Kvbeta1 and Kvbeta2 in rat brain, to investigate the expression of these beta-subunits in situ(8) . A major 38-kDa polypeptide and a minor 41-kDa polypeptide were detected in rat brain membrane fractions by immunoblot analysis. These two bands correspond closely to the predicted sizes of Kvbeta2 and Kvbeta1, respectively. Immunoprecipitation experiments showed that the major 38-kDa polypeptide is associated and colocalizes with Kv1.2 and Kv1.4, but not Kv2.1, in rat brain(8) , suggesting the selective interaction of K channel alpha- and beta-subunits.

As a first step toward understanding the molecular mechanisms that determine subunit composition of K channels, rat Kvbeta1 and Kvbeta2 were transfected either alone or together with K channel alpha-subunits into mammalian cells lacking these proteins. Kvbeta1 and Kvbeta2 exhibited selective interactions with Shaker- and Shal-related alpha-subunits but did not interact with Shab- and Shaw-related alpha-subunits. The interaction with alpha-subunits did not require the N-terminal domain necessary for the previously observed effects of some beta-subunits on alpha-subunit inactivation.


EXPERIMENTAL PROCEDURES

Materials

Dulbecco's modified Eagle's medium (DMEM) (^1)and methionine-free DMEM were from Life Technologies, Inc. Newborn calf serum was from Hyclone Laboratories. [S]Methionine (ExpreSS) was from DuPont NEN. Restriction and other enzymes were from Boehringer Mannheim. Horseradish peroxidase-conjugated secondary antibody was from Cappel (West Chester, PA). The enhanced chemiluminescence (ECL) reagents were from Amersham Corp. Pansorbin was from Calbiochem. Prestained molecular weight standards were from Sigma. All other reagents were from Sigma or Boehringer Mannheim.

Subunit-specific Antibodies

The rabbit polyclonal antibodies anti-beta and anti-Kv1.2 were produced and affinity purified essentially as described previously(8) . Anti-Kv1.1, Kv1.5, Kv1.6, and Kv4.2 antibodies were made against synthetic peptides listed below: Kv1.1, residues 458-476 CEEDMNNSIAHYRQANIRTG(9) ; Kv1.5, 586-602 CRRSLYALCLDTSRETDL(10) ; Kv1.6, 506-524 CRERRSYLPTPHRAYAEKR(10) ; and Kv4.2, 484-502 CLEKTTNHEFVDEQVFEES(11) . Antibodies were produced and purified essentially as described previously(8, 12) . Anti-Kv2.1 antibodies KC and pGEX-drk1 were produced as described previously(12) . Anti-Kv1.3 antibody T4(13) , generated by Dr. J. Douglass (Vollum Institute), was kindly provided by Dr. I. Levitan (Brandeis University). Anti-Kv3.1 antibody 602 (14) was kindly provided by Dr. T. Perney (Rutgers University).

Cloning of Kvbeta1 and Kvbeta2 cDNAs

We initially cloned a 475 bp fragment of Kvbeta1 cDNA by reverse transcriptase-PCR. Total adult rat brain RNA (1 µg) was reverse transcribed, and the resultant cDNA was subjected to PCR. Oligonucleotide primers B1-5-1 (21-mer, 5`-GGAATTCTGAGAGGACCTTGC-3`) and B1-3-1 (20-mer, 5`-TTCTTCCATGGGGGTGTTGC-3`) were used for 20 rounds of PCR. Amplified product was fractionated by an agarose gel, and a specific product of predicted size (642 bp) was isolated and subjected to 15 additional rounds of amplification using the same primers. The resultant product was isolated from an agarose gel, digested with EcoRI and HindIII, and cloned into Bluescript SK. Its identity as a fragment of Kvbeta1 cDNA was verified by sequencing. The clone was then used as a probe to screen a rat brain cDNA library (in ZAPII, kindly provided by Dr. T. Snutch (University of British Columbia)). From a total of 3.5 times 10^5 plaques, 9 Kvbeta1 clones were obtained. The identity of these clones was confirmed by restriction mapping and partial sequencing. One of the cDNA clones, pKB16, which contains the full-length Kvbeta1 coding region, 0.3 kbp of 5`-untranslated region, and 1.7 kbp of 3`-untranslated region, was used for further experiments.

As a result of this cDNA screening, we also obtained several Kvbeta2 clones. However, none of these clones contained full coding sequences. We again employed reverse transcriptase-PCR using the following primer set (5`-primer, B2-5 5`-CTGATCTAGATAAGTGAGGC-3`; 3`-primer, B2-3 5`-CTATCGATGACTTAGGATCTATAGTCC-3`), flanking the entire 1101-bp coding region of Kvbeta2, to obtain the coding region of Kvbeta2. After 25 rounds of PCR, a specific amplification product of the predicted size was obtained and subcloned into pRBG4 at the XbaI and ClaI sites. Eight cDNAs corresponding to the predicted size were identified by restriction mapping. One of these clones was analyzed by sequencing and was used as Kvbeta2/RBG4.

Construction of Mammalian Expression Vectors

cDNAs encoding alpha-subunits of voltage-gated K channel were kindly provided by investigators listed below: Kv1.1 (RBK1, (9) ), Dr. J. Adelman (Vollum Institute); Kv1.2 (rat RAK, (15) ), Dr. K. Rhodes (Wyeth Ayerst Research); Kv1.3 (Kv3, (10) ) and Kv1.6 (Kv2, (10) ), Dr. R. Swanson (Merck Research Labs); Kv1.5 (Kv1, (10) ), Dr. L. Kaczmarek (Yale Medical School); Kv2.1 (drk1, (16) ), Dr. R. Joho (University of Texas Southwestern Medical Center); and Kv3.1 (KV4, (17) ), Dr. T. Perney (Rutgers University). The Kv1.2 cDNA RAK was cloned from rat heart and differs at three nucleotides from rat brain cDNA (BK2, (18) ) kindly provided by Dr. D. McKinnon (SUNY, Stony Brook). We obtained similar results using either the RAT heart (RAK) or brain (BK2) cDNA; the data presented here were obtained with the RAK clone.

A 1.7-kbp EcoRI fragment of pKB16, which contains the entire coding region, was isolated and ligated into pRBG4 to generate Kvbeta1/RBG4. Expression vectors containing alpha-subunit cDNAs were constructed by cloning the respective coding regions into pRBG4 as follows. Kv1.1/RBG4 was generated by digesting Kv1.1/pS- (9) with PstI and HindIII, followed by ligation with PstI/HindIII-digested pRBG4. To generate Kv1.2 (rat RAK)/RBG4, a BglII fragment containing a coding region was isolated from rat RAK/pSP64T (15) and cloned into Bluescript SKBglII, a vector in the EcoRI site was changed to BglII, (^2)which was then digested with XbaI and HindIII, and the fragment was cloned into pRBG4. Kv1.3/RBG4 was constructed by digesting D541/pGemA (10) with EcoRI, followed by ligation into EcoRI-digested pRBG4. Kv1.5/RBG4 was generated by digesting clone D469 (10) with BstEII, followed by blunting with Klenow and ligation into EcoRV-digested pRBG4. Kv1.6/RBG4 was generated by digesting Kv2/pGEMA (10) with NotI, followed by blunting with Klenow, digestion with EcoRI, and ligation with EcoRV/EcoRI-digested pRBG4. The construction of Kv2.1/RBG4 was described previously(19) . Kv4.2/RBG4 was generated by digesting ratShal1/SK- with HincII and ligating the resultant fragment into EcoRV-digested pRBG4. The mammalian expression plasmid for Kv3.1 (in pRc/CMV) was obtained from Dr. T. Perney (Rutgers University).

Construction of the Kvbeta1DeltaN70 and Kvbeta2DeltaN22 Deletion Mutants

The Kvbeta1 deletion mutant Kvbeta1DeltaN70 was constructed from Kvbeta1/RBG4 by digestion with SmaI and ClaI, and the resultant fragment was ligated into EcoRV/ClaI-digested pRBG4. The Kvbeta2 deletion mutant Kvbeta2DeltaN22 was constructed from Kvbeta2/RBG4 by using the same procedure used to make Kvbeta1DeltaN70.

Expression and Analysis of K Channel alpha- and beta-Subunits

Procedures for COS-1 cell culture, DNA transfection, immunoblot analysis, and immunoprecipitation reactions were performed essentially as described in Shi et al.(19) with the following exceptions. For immunoblots, cells were extracted as described previously in 500 µl of lysis buffer, and the soluble lysate and insoluble pellet were separated by centrifugation in the microcentrifuge at 15,800 times g for 2 min. The supernatant (lysate) was diluted with an equal volume of 2 times reducing SDS sample buffer. For metabolic labeling in [S]methionine, cells grown on 60-mm tissue culture dishes were pre-incubated in methionine-free DMEM for 10 min at 37 °C followed by incubation in methionine-free DMEM containing 333 µCi/ml of [S]methionine at 37 °C for 2-4 h. Cells were then washed and extracted with 1 ml of lysis buffer. For immunoprecipitation reactions, 100 µl of lysate was used, and the resultant products were analyzed on 9% SDS-polyacrylamide gel electrophoresis and visualized by fluorography on Kodak BIOMAX film or by phosphorimaging (Molecular Dynamics).


RESULTS

Expression of beta-Subunits by Transient Transfection in COS-1 Cells

We have previously characterized the expression of beta-subunits in rat brain using an anti-beta-subunit antibody(8) . This antibody, raised against the conserved C-terminal region of Kvbeta1 and Kvbeta2 (and Kvbeta3), recognizes several polypeptides in rat brain, among these a predominant polypeptide species of 38 kDa, a polypeptide of 41 kDa, and minor species at 44 kDa (Fig. 1, lane 1). As a first step toward correlating these brain polypeptides with the recombinant beta-subunits, we expressed Kvbeta1 and Kvbeta2 cDNAs by transient transfection into COS-1 cells (19) and investigated the expressed polypeptides by immunoblots. Surprisingly, a minor immunoreactive polypeptide species of 44 kDa in rat brain membranes comigrates with the recombinant Kvbeta1 polypeptide (Fig. 1, lane 2). Comigration of the major beta-subunit immunoreactive polypeptide of 38 kDa with recombinant Kvbeta2 (Fig. 1, lane 3) is consistent with our previous proposal (8) that this abundant brain polypeptide is in fact Kvbeta2. Similar electrophoretic mobilities for these recombinant beta-subunits are obtained in two other mammalian cell lines (HEK293, PC12; not shown), suggesting that cell type-specific post-translational modifications do not contribute significantly to the mobility of beta-subunit polypeptides. This suggests, but does yet not prove, that the prominent 41-kDa immunoreactive band in rat brain is not Kvbeta1 and that Kvbeta1 apparently corresponds to the 44-kDa polypeptide.


Figure 1: Immunoblot analysis of beta-subunits in rat brain membranes and in transfected COS-1 cells. Crude rat brain membranes (75 µg, lane 1) and the detergent extracts of COS-1 cells transfected with Kvbeta1/RBG4 (lane 2), Kvbeta2/RBG4 (lane 3), or mock-transfected cells (lane 4) were fractionated on a 9% SDS gel, transferred to nitrocellulose, and the resultant immunoblot probed with anti-beta subunit antibody. Signals were visualized by autofluorography using ECL. Numbers on right refer to mobility of prestained molecular weight standards.



Specific Association of Kvbeta1 and Kvbeta2 with Kv1.2

To study the selectivity of alpha/beta-subunit interaction, we undertook a biochemical approach utilizing co-immunoprecipitation from cotransfected COS-1 cells. Except where explicitly stated otherwise, all immunoprecipitation reactions were performed under conditions designed to maintain subunit association, resulting in some nonspecific background, even in reactions performed in the absence of antibody. Initially, we focused on beta-subunit interaction with Kv1.2, based on previous studies in brain(3, 8) . Fig. 2shows a fluorographic image of immunoprecipitation products fractionated on an SDS gel. Kv1.2-, Kvbeta1-, and Kvbeta2-transfected cells express 65-, 44-, and 38-kDa proteins, respectively (Fig. 2), and in each case subunit-specific antibodies show no detectable cross-reactivity to heterologous samples. As expected, Kvbeta1 and Kvbeta2 are immunoprecipitated with the pan-beta antibody from Kv1.2/Kvbeta1- or Kv1.2/Kvbeta2-cotransfected cells (Fig. 2). Both beta-subunits could also be co-immunoprecipitated with the anti-Kv1.2 antibody. The presence of Kv1.2/Kvbeta interaction was confirmed by reciprocal co-immunoprecipitation reactions by the presence of Kv1.2 in the beta-subunit immunoprecipitation products.


Figure 2: Association of Kvbeta1 and Kvbeta2 with Kv1.2 in co-expressing COS-1 cells. Kv1.2 and beta-subunits were expressed either alone or co-expressed with one another. Cells were labeled with [S]methionine for 2 h, harvested in lysis buffer, and the lysates were subjected to immunoprecipitation with anti-Kv1.2 (``alpha'' lanes) or anti-beta (``beta'' lanes) antibody. The combinations of Kv1.2 and beta-subunit cDNAs are shown above the lanes. Numbers on left refer to mobility of prestained molecular weight standards.



Addition of a denaturing agent, such as the detergents SDS and deoxycholate, should affect polypeptide folding and disrupt the noncovalent protein-protein interactions typical of most multi-subunit membrane protein complexes(20) . To test if K channel subunit association was through similar noncovalent interactions, immunoprecipitation reactions were performed in the presence of such denaturing agents. The coprecipitation of Kv1.2 with Kvbeta2 could be disrupted by the addition of 0.2% SDS and 0.5% sodium deoxycholate during the immunoprecipitation reactions; this treatment has no effect on the direct immunoprecipitation of the subunits themselves (Fig. 3A). Similar results were obtained for Kv1.2-Kvbeta1 interaction (not shown). Thus, K channel alpha/beta-subunit interaction has similar sensitivity to denaturing detergents as exhibited for other multisubunit membrane protein complexes(20) .


Figure 3: A, effect of SDS addition to immunoprecipitation reactions. Cells were transfected with Kv1.2 and Kvbeta2. Cells were labeled with [S]methionine for 2 h, harvested in lysis buffer, and the lysates were subjected to immunoprecipitation with anti-Kv1.2 (``alpha'' lanes) or anti-beta (``beta'' lanes) antibody or without antibody (``-'' lanes). Immunoprecipitation reactions were carried out under the presence (right panel) or absence (left panel) of 0.2% SDS and 0.5% sodium deoxycholate. Numbers on left refer to mobility of prestained molecular weight standards. B, immunoprecipitation from the mixed-cell lysate of individually transfected dishes of cells. COS-1 cells individually transfected with Kv1.2, Kvbeta1, or Kvbeta2 were mixed and lysed, and the lysates were subjected to immunoprecipitation. The combinations of the singly transfected cells used for immunoprecipitation are indicated at the top of the lanes labeled ``mix''. Lanes labeled ``co'' show the results obtained from cotransfected cells expressing the same alpha/beta-subunit combination. The immunoprecipitation reactions were performed with anti-Kv1.2 (``alpha'' lanes) or anti-beta (``beta'' lanes) or without antibody (``-'' lanes). Numbers on left refer to mobility of prestained molecular weight standards.



To test whether co-expression within the same cell is necessary for subunit interaction, individually transfected dishes of COS-1 cells expressing either Kv1.2 or Kvbeta2 were harvested. The cells were then pooled, and the pooled mixture of cells was extracted under standard conditions. The resultant lysates were then subjected to immunoprecipitation with subunit-specific antibodies. These experiments yielded no co-immunoprecipitation of alpha- and beta-subunits above background (no antibody lanes), showing that co-expression within the same cell is necessary for subunit interaction (Fig. 3B).

Previous studies had shown that deletion of the N terminus of Kvbeta1 destroyed its ability to modulate inactivation(4) . To test whether this was simply due to a lack of interaction, an N-terminal truncation mutant, Kvbeta1DeltaN70, which lacks amino acids 1-70, was co-expressed with Kv1.2. As shown in Fig. 4A, Kvbeta1DeltaN70 can be efficiently co-immunoprecipitated with anti-Kv1.2 antibody and vice-versa. Thus, removal of the domain necessary for Kvbeta1-mediated modulation of inactivation does not disrupt alpha/beta-subunit interaction, showing that the loss of the ability of such mutants to modulate inactivation is not due to an inability to interact with alpha-subunits. A similar N-terminal deletion of Kvbeta2 (Kvbeta2DeltaN22) also exhibited interaction with Kv1.2 that was indistinguishable from wild-type Kvbeta2 (Fig. 4B). These data indicate that the N-terminal domains of beta-subunits are not necessary for the interaction with alpha-subunits and that the interaction domain lies somewhere else in the beta-subunit sequence.


Figure 4: Association of N-terminal truncation mutants of Kvbeta1 and Kvbeta2 with Kv1.2. A, Kv1.2 and Kvbeta1DeltaN70 were co-expressed in COS-1 cells, labeled with [S]methionine for 2 h, and the lysate was examined by immunoprecipitation with anti-beta (``beta'' lane) or anti-Kv1.2 (``alpha'' lane) antibody. B, Kv1.2 and Kvbeta2DeltaN22 were co-expressed in COS-1 cells, labeled with [S]methionine for 2 h, and the lysate was examined by immunoprecipitation with anti-beta (``beta'' lane) or anti-Kv1.2 (``alpha'' lane) antibody. Numbers on left refer to mobility of prestained molecular weight standards.



Selective Association of Kvbeta1 and Kvbeta2 with alpha-Subunits

To investigate the selectivity of alpha/beta-subunit interaction, co-immunoprecipitation from cells co-expressing pairwise combinations of recombinant mammalian alpha-subunits and Kvbeta1 and Kvbeta2 was performed. Control experiments, as detailed above, were performed for each set of alpha/beta-subunit combinations. However, due to space limitations, only the relevant co-immunoprecipitation reactions are presented here.

Kv1 Subfamily

Five members of the mammalian Shaker-related (Kv1) subfamily were tested for interaction with Kvbeta1 and Kvbeta2. All of the Kv1 family members tested (Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv1.6) exhibit direct and specific interaction with both Kvbeta1 and Kvbeta2, as evidenced by reciprocal co-immunoprecipitation (Fig. 5). However, distinctions are apparent in the extent of co-immunoprecipitation among the specific pairwise combinations. Kv1.3 and Kv1.6 are similar to Kv1.2, in that high levels of co-immunoprecipitation of both the alpha- and beta-subunits are observed in reactions using either anti-alpha-subunit or anti-beta-subunit antibody. Kv1.1 and Kv1.5, however, show lower than expected levels of coprecipitated alpha-subunits in the anti-beta-subunit immunoprecipitation reactions, perhaps due to an overabundance of beta-subunits such that only a small fraction of the large total beta-subunit pool is associated with the small alpha-subunit pool. However, it is not possible to determine from these types of experiments whether these differences reflect quantitative differences in alpha/beta-subunit association. Control experiments on singly transfected cells expressing alpha-subunits alone show no detectable immunoprecipitation with anti-beta-subunit antibodies. This verifies that the low levels of Kv1.1 and Kv1.5 seen in anti-beta-subunit immunoprecipitation reactions performed on cells co-expressing alpha- and beta-subunits are specific and significant.


Figure 5: Association of Kvbeta1 and Kvbeta2 with Shaker-related alpha-subunits. Interactions between various alpha- and beta-subunits were examined by immunoprecipitation. Panels show the results of co-immunoprecipitation reactions obtained from COS-1 cells co-expressing Kvbeta1 and Kvbeta2 with Kv1.1, Kv1.3, Kv1.5, or Kv1.6 alpha-subunits. The combinations of alpha- and beta-subunit cDNAs are shown above the lanes. Cells were labeled with [S]methionine for 2 h, and the lysates were examined by immunoprecipitation with anti-beta (``beta'' lanes) or antibody specific to the alpha-subunit shown above the each panel (``alpha'' lanes). Numbers on left refer to mobility of prestained molecular weight standards.



Kv2 and Kv3 Subfamily

Analysis of cells cotransfected with Shab-related Kv2.1 and either Kvbeta1 or Kvbeta2 show no coprecipitation by anti-beta subunit antibody (Fig. 6A). Low levels of Kvbeta1 and Kvbeta2 are seen in immunoprecipitation reactions performed with the anti-alpha-subunit antibody; however, comparable levels are observed in similar immunoprecipitation reactions performed on cells expressing beta-subunits alone (not shown), indicating that these products are due to minor cross-reactivity of the anti-Kv2.1 antibody to these beta-subunits and not to alpha/beta-subunit interaction. Similar nonspecific immunoprecipitation of low levels of beta-subunits was also seen in immunoprecipitation reactions performed on cotransfected cells in the absence of antibody (not shown). The addition of denaturing agents, SDS and deoxycholate, during the immunoprecipitation reactions shows that this treatment has no or very weak effect on the relatively low but detectable level of co-immunoprecipitation (Fig. 6B). Taken together, these data indicate that the observed co-immunoprecipitation is not due to specific noncovalent interactions between Kv2.1 and Kvbeta2, as these sorts of intermolecular associations are typically disrupted by denaturing agents (see Fig. 3A) but is due to low levels of antibody cross-reactivity or other nonspecific precipitation. When these beta-subunits were co-expressed with the mammalian Shaw homolog Kv3.1, no detectable co-immunoprecipitation was observed (Fig. 6C), although strong subunit-specific immunoprecipitation was observed.


Figure 6: Association of Kvbeta1 and Kvbeta2 with Shab- and Shaw-related alpha-subunits. Panels show the results of co-immunoprecipitation reactions obtained from COS-1 cells co-expressing Kvbeta1 and Kvbeta2 with Shab-related (Kv2.1, panel A) or Shaw-related (Kv3.1, panel C) alpha-subunits. In panel B, lysates from COS-1 cells transfected with Kv2.1 and Kvbeta2 were subjected to immunoprecipitation under the presence (right panel) or absence (left panel) of 0.2% SDS and 0.5% sodium deoxycholate. Cells were labeled with [S]methionine for 2 h, and the lysates were examined by immunoprecipitation with anti-beta (``beta'' lanes), anti-Kv2.1 (pGEX-drk1, ``alpha'' lanes in A and B), or anti-Kv3.1 (``alpha'' lanes in C). Numbers on left refer to mobility of prestained molecular weight standards.



Kv4 Subfamily

Strong reciprocal co-immunoprecipitation was observed between both Kvbeta1 and Kvbeta2 and the mammalian Shal homolog Kv4.2 (Fig. 7A). The interaction of Kvbeta2 and Kv4.2 is relatively resistant to treatment with the denaturing detergent SDS (SDS treatment) in that the co-immunoprecipitation is not disrupted by the addition of SDS at concentrations less than 0.6% (Fig. 7B). This is distinct from the characteristics of the interaction of Kv1.2 with Kvbeta1 and Kvbeta2, where interaction is partially disrupted by the addition of SDS to only 0.2%, with complete disruption observed at 0.4% SDS (Fig. 7B).


Figure 7: A, association of Kvbeta1 and Kvbeta2 with Shal-related alpha-subunit. Kv4.2 and Kvbeta1 (+beta1) or Kvbeta2 (+beta2) were co-expressed in COS-1 cells. Cells were labeled with [S]methionine for 2 h, and the lysate was examined by immunoprecipitation with anti-beta (``beta'' lanes) or anti-Kv4.2 (``alpha'' lanes) antibody. Numbers on left refer to mobility of prestained molecular weight standards. B, effect of SDS addition on the co-immunoprecipitation of Kv1.2/Kvbeta2 and Kv4.2/Kvbeta2. COS-1 cells were transfected with Kv1.2 and Kvbeta2 or Kv4.2 and Kvbeta2 and labeled with [S]methionine for 2 h. The immunoprecipitation reactions were carried out under the presence of various concentrations of SDS indicated above the lanes. Anti-beta antibody was used for precipitation. Numbers on left refer to mobility of prestained molecular weight standards.




DISCUSSION

Our previous study using an antibody against a sequence conserved in both Kvbeta1 and Kvbeta2 revealed the existence of multiple immunoreactive beta-subunits in rat brain(8) . Here, analysis of transfected cells expressing recombinant Kvbeta2 and Kvbeta1 reveals that a minor 44-kDa rat brain beta-subunit comigrates with Kvbeta1, while the major 38-kDa beta-subunit comigrates with Kvbeta2. The other immunoreactive beta-subunit at 41 kDa, which is recognized by the beta-subunit antibody, is apparently neither Kvbeta1 nor Kvbeta2 and suggests the existence of an additional, as yet uncharacterized member of the beta-subunit gene family in rat brain. Recent cloning of a partial cDNA for a rat Kvbeta3 beta-subunit, which shares the same nucleotide sequence with Kvbeta1 except for its unique N-terminal region and is predicted to encode a polypeptide of 45 kDa, strongly suggests the presence of at least one alternatively spliced product of the Kvbeta1 gene(5) . Studies with subtype-specific antibodies will allow for the eventual unequivocal identification and localization of each of the individual components of the beta-subunit pool in brain. Moreover, comprehensive molecular analysis of the beta-subunit gene family will lead to the identification of other beta-subunits, for instance, those associated with Shab- (see (12) ), Shaw-, and Shal- (21) related K channels.

We found that Kvbeta1 and Kvbeta2 expressed in COS-1 cells could associate with all five of the Shaker-related subfamily members tested (Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6), as well as with the Shal-related Kv4.2. The ratio of the amount of co-immunoprecipitation seen on anti-alpha and anti-beta lanes exhibited some variation among the different alpha/beta pairwise combinations. This discrepancy in the extent of reciprocity of co-immunoprecipitation could be due to the relatively low efficiency of immunoprecipitation with anti-beta antibody due to the high expression level of beta-subunit in the cotransfected cells. Kv2.1 and Kv3.1 exhibited no detectable co-immunoprecipitation, suggesting these two alpha-subunits are unable to interact with Kvbeta1 and Kvbeta2. Subcellular localization of Kv2.1 and beta-subunits in transfected cells is consistent with this model in that immunofluorescence staining of cells co-expressing Kv2.1 and Kvbeta2 shows no overlap of alpha- and beta-subunit staining, while cells co-expressing Kv1.2 and Kvbeta2 with Kvbeta2 show extensive overlap throughout the cells. (^3)

Our results provide direct biochemical evidence for selective interaction of K channel beta-subunits with only a subset of the alpha-subunit gene family, have greatly expanded the initial observations of Rettig et al.(4) who showed that Kv1.1 and Kv1.4 interact functionally with Kvbeta1 in oocytes(4) , and provide the evidence for a direct, noncovalent interaction between alpha- and beta-subunits. These results also confirm and extend our previous studies of rat brain alpha/beta-subunit association in situ, where we found that neuronal beta-subunits could be coprecipitated with rat brain Kv1.2 and Kv1.4 but not with Kv2.1(8) . A detailed characterization of purified bovine brain dendrotoxin acceptors, which were later found to contain Kvbeta1 and Kvbeta2(3) , showed that these K channel complexes contain Kv1.1, Kv1.2, Kv1.4, and Kv1.6 (22) . Our findings provide a first step toward understanding the molecular determinants of alpha/beta-subunit interaction by showing that the subunit selectivity observed in rat brain can be recapitulated in transfected cell lines, indicating that selectivity is mainly determined by the primary structure of the interacting subunits.

The voltage-gated K channel alpha-subunit genes segregate into four subfamilies based on the similarity of primary structure of each member(23) . As discussed above, our results show that Kvbeta1 and Kvbeta2 interaction seemed to be restricted to Shaker- and Shal-related subfamilies. Interestingly, proposed phylogenetic trees place the Shaker (Kv1) and Shal (Kv4) subfamilies on one major branch, while Shab (Kv2) and Shaw (Kv3) members are placed on a separate branch(1, 24) . Thus, the ability to interact with Kvbeta1 and Kvbeta2 appears to reside in the relatedness of their primary sequences as evidenced by their phylogenetic grouping and allows for the design of structure-function analyses aimed at defining the domains of alpha-subunits mediating alpha/beta-subunit interaction. In the case of voltage-sensitive Ca channel alpha/beta-subunit interaction, the beta-subunit binds to a conserved cytoplasmic motif in the alpha(1)-subunit(25) . Taken together with the fact that K channel beta-subunits are also cytoplasmic proteins, it is likely that the interaction domain on K channel alpha-subunits is present on a cytoplasmic domain.

No distinct domain of alpha-subunits stands out as a clear candidate for mediating interaction with beta-subunits based simply on the positive interaction of both Kv1 and Kv4 family members. However, our experiments using SDS treatment to disrupt alpha/beta-subunit interaction imply that the interaction of beta-subunits with Kv1.2 and Kv4.2 are somewhat distinct. In addition, only Kv1 and not Kv2, Kv3, or Kv4 subfamily alpha-subunits have been found associated with Kvbeta1 and Kvbeta2 in rat brain in situ(8) . (^4)Together, these data may imply that the only physiologically relevant subunit interactions are between Kv1 (Shaker-related) alpha-subunits and Kvbeta1 and Kvbeta2. Using this assumption, a conserved N-terminal, presumably cytoplasmic domain of about 130 amino acids is striking in that it is highly conserved among Kv1 alpha-subunits but not among members of the other (Kv2, Kv3, and Kv4) subfamily members. This highly conserved region, known as the ``T1'' (26) or ``NAB'' (27) domain, is thought to be important in mediating efficient alpha/alpha-subunit interaction(26, 27, 28) . This may raise the interesting scenario whereby both alpha/alpha- and alpha/beta-subunit interactions are mediated through similar domains. Extensive mutational analysis of alpha-subunit proteins will lead to the elucidation of the specific beta-subunit binding region.


FOOTNOTES

*
This work was supported by the Central Nervous System Division, Wyeth-Ayerst Research, by the Center for Biotechnology at Stony Brook, funded by the New York State Science and Technology Foundation, by grants from the Council for Tobacco Research, and by National Institutes of Health Grant NS34383 (to J. S. T.); this work was done during the tenure of an Established Investigatorship from the American Heart Association (to J. S. T.). 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 should be addressed. Tel.: 516-632-9171; Fax: 516-632-8575; trimmer{at}pofvax.sunysb.edu.

(^1)
The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; bp, base pair(s); kbp, kilobase pair(s).

(^2)
J. S. Trimmer, unpublished data.

(^3)
K. Nakahira, G. Shi, and J. S. Trimmer, unpublished data.

(^4)
K. J. Rhodes, Z. Bekele-Arcuri, M. M. Monoghan, and J. S. Trimmer, manuscript in preparation.


ACKNOWLEDGEMENTS

-We thank Dr. R. Haltiwanger for critically reviewing this manuscript and Zewditu Bekele-Arcuri and Tina Matos for expert technical assistance. We are particularly grateful to each of the investigators listed above for generously providing cDNA clones and antibodies.


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