Kvbeta 2 Inhibits the Kvbeta 1-mediated Inactivation of K+ Channels in Transfected Mammalian Cells*

(Received for publication, January 13, 1997, and in revised form, February 14, 1997)

Jia Xu and Min Li Dagger §

From the Department of Physiology and Dagger  Department of Neuroscience, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Cloned auxiliary beta -subunits (e.g. Kvbeta 1) modulate the kinetic properties of the pore-forming alpha -subunits of a subset of Shaker-like potassium channels. Coexpression of the alpha -subunit and Kvbeta 2, however, induces little change in channel properties. Since more than one beta -subunit has been found in individual K+ channel complexes and expression patterns of different beta -subunits overlap in vivo, it is important to test the possible physical and/or functional interaction(s) between different beta -subunits. In this report, we show that both Kvbeta 2 and Kvbeta 1 recognize the same region on the pore-forming alpha -subunits of the Kv1 Shaker-like potassium channels. In the absence of alpha -subunits the Kvbeta 2 polypeptide interacts with additional beta -subunit(s) to form either a homomultimer with Kvbeta 2 or a heteromultimer with Kvbeta 1. When coexpressing alpha -subunits and Kvbeta 1 in the presence of Kvbeta 2, we find that Kvbeta 2 is capable of inhibiting the Kvbeta 1-mediated inactivation. Using deletion analysis, we have localized the minimal interaction region that is sufficient for Kvbeta 2 to associate with both alpha -subunits and Kvbeta 1. This mapped minimal interaction region is necessary and sufficient for inhibiting the Kvbeta 1-mediated inactivation, consistent with the notion that the inhibitory activity of Kvbeta 2 results from the coassembly of Kvbeta 2 with compatible alpha -subunits and possibly with Kvbeta 1. Together, these results provide biochemical evidence that Kvbeta 2 may profoundly alter the inactivation activity of another beta -subunit by either differential subunit assembly or by competing for binding sites on alpha -subunits, which indicates that Kvbeta 2 is capable of serving as an important determinant in regulating the kinetic properties of K+ currents.


INTRODUCTION

The heterogeneity of voltage-sensitive potassium currents present in excitable and nonexcitable cells is essential for diverse biological functions (1-4). In addition to the large number of genes encoding the channel subunits and posttranslational modulations of channel protein, the diversity of potassium channels is further enhanced by the mix-and-match assembly of different subunits (5, 6). Within the large family of Shaker-like potassium channels, the selective subunit assembly includes heteromultimer formation of distinct pore-forming alpha -subunits and/or assembly of different kinds of subunits such as the alpha -subunits and hydrophilic cytoplasmic beta -subunit(s) (7, 8). Together, these give rise to the vast heterogeneity of K+ currents. Changes in expression of a given subunit may alter the composition of heteromultimers in vivo, which would allow a cell to tune its K+ current system(s) during development and in response to changes in the cellular environment.

There are more than 60 cloned genes encoding functional Shaker-like alpha -subunits, which have been divided into several subfamilies. Among them, subunits in the Kv1 to Kv5 subfamily are capable of functional homomeric channels in heterologous systems, such as Xenopus oocytes (9-13). Four alpha -subunits within a given subfamily can form a functional channel either as a homotetramer or a heterotetramer (14, 15). In the case of auxiliary subunits in animals, four genes encoding beta -subunits for Shaker-like potassium channels have been well characterized: Kvbeta 1, Kvbeta 2, Kvbeta 3 (which has now been suggested to be a splice variant of Kvbeta 1), and Hk (7, 8, 16-20). These beta -subunits share at least 85% amino acid sequence identity in their COOH-terminal core regions, but differ significantly in length and sequence of the remaining NH2-terminal regions. Despite the remarkable sequence similarity among different beta -subunits, their functional effects are quite different. For example, coexpression of beta -subunits with certain alpha -subunits in Xenopus oocytes induces pronounced alterations in channel kinetic properties, most noticeably acceleration of fast inactivation by either Kvbeta 1 or Kvbeta 3 (8, 17, 18-20). Kvbeta 2, on the other hand, binds to alpha -subunits, such as Kv1.2 (or RCK5) (21). However, it has little effect on inactivation of alpha -subunits such as Kv1.2 (RCK5) (7, 8, 22, 23). Recent data have shown that Kvbeta 2 is capable of increasing the surface expression of certain K+ channels in transfected cells (24).

Biochemical evidence has indicated that there are more than one beta -subunit present in each K+ channel complex (21). Given that cloned beta -subunits have different modulatory effects on alpha -subunits, it would be interesting to test whether different beta -subunits can interact with each other, which could be an important mechanism to increase the diversity of potassium currents. To test this hypothesis, we have used the yeast two-hybrid system to study the interaction specificity of Kvbeta 2 with various alpha - and beta -subunits. The functional consequences of heteromeric alpha -beta and beta -beta interactions were evaluated by electrophysiological analyses.


MATERIALS AND METHODS

Vector Construction and Expression

Plasmid vector construction was performed according to standard recombinant DNA techniques (26). The vectors that express partial cDNA fragments were constructed by a high fidelity polymerase chain reaction cloning strategy according to the procedures described by Li et al. (27). The oligonucleotides used are listed in Table I. In yeast, the expression of different fusion proteins of alpha -subunits, Kvbeta 1, and Kvbeta 2 was carried out by inserting the corresponding cDNA fragments into the SmaI/NotI, SalI/NotI, or BglII/NotI sites of pPC97 and pPC86 vectors (28-31). Construction of tagged Kvbeta 1, Kvbeta 2, and Kvbeta 2 mutants was carried out by fusing the coding fragment with a peptide which represents a heart muscle kinase recognition sequence and the 12CA5 monoclonal epitope (PYDVPDYASL), at the end of the coding sequences before the stop codon (28). Transient expression and immunodetection of potassium channel subunits were performed according to our published protocol (28, 51).

Table I. Sequences of oligonucleotide primers


Oligonucleotides Sequencesa Gene Amino acid position

ML1005: GCACCATGGAGTCGACA Kvbeta 2 1
ML1006: CAGGCGGCCGC Kvbeta 2 367
ML1015: CAGGTCGACTGAATTC Kvbeta 2 39
ML1021: CAGGAATTCAAGATCTCA Kvbeta 1 1
ML1022: CAGAGATCTC Kvbeta 1 73
ML1023: CAGAGATCTT Kvbeta 1 131
ML1024: CAGAGATCT Kvbeta 1 196
ML1025: CAGGCGGCCGCTA Kvbeta 1 289
ML1026: CAGGCGGCCGCTA Kvbeta 1 346
ML1027: CAGGCGGCCGCTA Kvbeta 1 401
ML1031: CAGGAATTCGAAGCA Kvbeta 1 1
ML1032: CAGGAATTC Kvbeta 1 401
ML1044: CAGGAATTCGGCCCC Kvbeta 2 1
ML1045: CAGGAATTC Kvbeta 2 367
ML1069: CAGGAATTCGGCCCCATG Kvbeta 2 39
ML1079: CAGGAATTC Kvbeta 2 316

a The underlined sequence represents coding sequence or complementary to coding sequence of the indicated gene.

Methods for the Yeast Two-hybrid System

The procedures were performed according to our published protocol (28, 29) using HF7c yeast strain (MATalpha ura3-52 his-200 ade 2-101 lys2-801 trp1-901 leu2-3, 112 gal4-542 gal80-538 LYS2::GAL1UAS-GAL1TATA-HIS3 URA3::GAL4 17mer(x3)-CyclTATA-lacZ) as host cells (25).

Whole-cell Patch Clamp Recording

Whole-cell voltage clamp recordings were carried out according to the published protocol (28, 33). The liquid junction potential was calculated to be 7.2 mV using JPCalc software (34) and corrected from the holding potential. Typically, the cell was held at -87 mV, and the holding voltage was then jumped from this potential up to a test potential of +53 mV in 10-mV increments for 300 ms. Current data were filtered at 1 kHz, digitized at 100-µs intervals. Data analysis was done using clampfit software (pCLAMP6, Axon Instrument, Foster City, CA).

Statistical Analysis

A standard formula to compare two proportions was used to determine the statistical significance of different pairs of sample sets (35). We have used tau  = 64 ms as the cutoff to separate populations with or without fast inactivation. In this analysis, the one-sided z test statistics were calculated using the following formula z =  theta 1-theta 2 /{theta p(1-theta p)[1/n1 + (1/n2)]}0.5, where theta 1 and theta 2 are sample proportions that showed fast inactivation of each given group, theta p is the weighted average of the sample proportions, and n1 and n2 are sample sizes.


RESULTS

Differential Modulation on alpha -Subunits by Kvbeta 1 and Kvbeta 2

The interaction between alpha -subunits and Kvbeta 1 has been studied in more detail. In particular, the alpha -beta complex is assembled, at least in part, by the association of the conserved core regions in Kvbeta 1 with NABKv1 of alpha -subunits, a critical assembly motif located in the hydrophilic NH2-terminal domains (28, 29, 36). The formation of an alpha -beta complex presumably recruits the inactivation particle of Kvbeta 1 close to the "receptor" site, thereby either accelerating the rate of inactivation of alpha -subunits of Kv1.4 (RCK4) and ShB (or H4) or inducing inactivation of compatible alpha -subunits which lack intrinsic fast inactivation, such as ShBDelta (6-46) (28, 36). Because the formation of the alpha -Kvbeta 1 complexes is subfamily-specific, i.e. Kvbeta 1 binds only to the NH2-terminal domains of Kv1 alpha -subunits (28), this allows Kvbeta 1 to selectively modulate a subset of alpha -subunits.

To investigate whether Kvbeta 2 alters the electrophysiological properties of alpha -subunits in transfected mammalian cells, we have constructed two plasmids that express either Kvbeta 1 or Kvbeta 2 with the 12CA5 monoclonal antibody tag fused at the COOH terminus of each coding sequence (see "Materials and Methods"). Both plasmids use the Kvbeta 1 5'-untranslated sequence. Thus, the two expression vectors are identical except for the amino acid coding sequence. Experiments utilizing these constructs permit better comparison of Kvbeta 1 and Kvbeta 2 expression and their ability to modulate alpha -subunits. By transient transfection in COS cells, we functionally expressed ShBDelta (6-46), a mutated ShB potassium channel that lacks the inactivation gate (37, 38). The Kvbeta 2 effects on this alpha -subunit were studied by whole-cell voltage clamp recording. Fig. 1A shows a series of traces obtained by stepping up from a holding potential of -87 mV to a final test potential of +33 mV in 20-mV increments. The recorded cells were transfected with ShBDelta (6-46) alone (upper panel), ShBDelta (6-46) in the presence of either Kvbeta 1 (middle panel), or Kvbeta 2 (bottom panel). In contrast to the ShBDelta (6-46) + Kvbeta 1 cotransfection in which we observed the Kvbeta 1-mediated inactivation (Fig. 1A, middle panel), expression of ShBDelta (6-46) in the presence of Kvbeta 2 resulted in no detectable changes of the fast inactivation properties (Fig. 1A, bottom panel). When traces were averaged within the group of ShBDelta (6-46) + Kvbeta 2 (n = 12) or ShBDelta (6-46) alone (n = 17), we observed little variations of inactivation properties between these two groups of recorded cells (Fig. 1B). To examine the protein expression of Kvbeta 2 in the experiments, total cell lysates from the transfected cells were separated by SDS-polyacrylamide gel electrophoresis. The expression of Kvbeta 1 and Kvbeta 2 was detected by immunoblot using the 12CA5 monoclonal antibody (mAb12CA5). Indeed, Kvbeta 2 was found to express in the transfected COS cells and exhibited higher expression as compared with that of Kvbeta 1 (Fig. 1C, lanes 1 and 2). The higher expression of Kvbeta 2 has been reproducible in multiple transfection experiments.1 The strong mAb12CA5 binding signal indicates that the failure of Kvbeta 2 to modulate N-type fast inactivation was not due to the lower protein expression of Kvbeta 2. Thus, Kvbeta 2 differs from Kvbeta 1 and by itself fails to induce the fast inactivation of the ShB alpha -subunit.


Fig. 1. Differential modulation of Kv1 alpha -subunits by Kvbeta 1 and Kvbeta 2. A, K+ currents recorded by whole-cell voltage clamp. COS cells transfected with different combinations of plasmids: ShBDelta (6-46) only (n = 17, top), ShBDelta (6-46) + Kvbeta 1(n = 15, middle), or ShBDelta (6-46) + Kvbeta 2 (n = 12, bottom). Plasmid inputs in transfections were 3 µg for ShBDelta (6-46) and 18 µg for beta -subunits. A plasmid (2 µg) encoding the CD4 antigen is included in all COS transfections for whole-cell voltage clamp recording. Typical current responses were obtained by stepping up holding potential from -87 mV to a final test potential of +33 mV in 20-mV increments, and one cell from each group is shown. Scale bars, 5 nA and 100 ms. B, averaged K+ current responses of ShBDelta (6-46) in the presence or absence of Kvbeta 2. Current responses to a voltage step from -87 mV to +33 mV were normalized according to the peak current and averaged within the group. The control trace shows the averaged current response recorded from 17 ShBDelta (6-46)-transfected COS cells. The second trace (indicated by the arrow) illustrates the averaged current response obtained from 12 COS cells transfected with ShBDelta (6-46) + Kvbeta 2. The time scale is identical to that in A. C, expression of Kvbeta 1 and Kvbeta 2 polypeptides in COS cells detected by immunoblot analysis. An aliquot of the transfected COS cells from each of the above transfections was collected. Total cell lysates were prepared according to a standard protocol (Ref. 28; see "Materials and Methods"). Protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The 12CA5-tagged Kvbeta 1 and Kvbeta 2 polypeptides (as indicated on the side) were detected by an affinity-purified 12CA5 monoclonal antibody. The lower panel is a non-12CA5 signal endogenous to COS cells, indicating that comparable amounts of protein were loaded in each lane.
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The Subfamily-specific Association of Kvbeta 2 with Kv1 alpha -Subunits

Biochemical characterization supports direct physical interaction between Kvbeta 2 and Kv1.2 (RCK5) (7, 21) as well as Kv1.4 (RCK4) (39). However, the existing results for Kvbeta 2 binding specificity and region(s) involved are not conclusive (Ref. 23, also see "Discussion"). In addition, there is no information on whether Kvbeta 2 binds to ShB. To test the association between Kvbeta 2 and Kv1 alpha -subunits, we expressed various regions of alpha -subunits and Kvbeta 2 in yeast and used the yeast two-hybrid system to study the potential interaction(s) (30, 31). In the case of Kvbeta 1, it has been found that the NH2-terminal domains of the Kv1 alpha -subunits are involved in the alpha -Kvbeta 1 interaction (28, 36). Because Kvbeta 1 and Kvbeta 2 share considerable sequence homology, we first tested the potential interaction of Kvbeta 2 with the cytoplasmic regions of the Kv1.4, an alpha -subunit which has been found to interact with Kvbeta 2 (39). The truncated cytoplasmic fragments, i.e. the NH2-terminal domain (aa2 1-306) and COOH-terminal domain (aa 566-651), were expressed individually with Kvbeta 2 as GAL4 fusion proteins. If Kvbeta 2 interacts with one or both truncated Kv1.4 fragments, the resultant interaction(s) should confer the ability of the yeast transformants to grow on synthetic medium lacking histidine. Fig. 2A shows that when Kvbeta 2 was expressed alone either as a fusion protein of the GAL4 DNA binding domain (GAL4-DB) or that of the GAL4 transcription activation domain (GAL4-TA), the yeast transformants grew on double selection medium supplemented with histidine, indicating that they carry both plasmids (Fig. 2A, numbers 1 and 2, middle left panel). When the same number of transformants were tested to grow on the triple selection medium lacking histidine, they showed no growth (Fig. 2A, numbers 1 and 2, lower left panel). This indicates that Kvbeta 2 itself does not exert any endogenous activity that permits the yeast transformants to grow on the selection medium. By contrast, the coexpression of Kvbeta 2 and the NH2-terminal domain (Fig. 2A, number 3), not the COOH-terminal domain (Fig. 2A, number 4) of Kv1.4, resulted in growth on the selection medium lacking histidine. Consistent results have been obtained using a beta -galactosidase assay (data not shown). Thus, similar to Kvbeta 1, Kvbeta 2 interacts with the NH2-terminal domain of the Kv1.4 alpha -subunit.


Fig. 2. Subfamily-specific interaction of Kvbeta 2 with the NH2-terminal domains of the Kv1 alpha -subunits. HF7c yeast cells were transformed by different pairwise combinations of the two-hybrid constructs that express fusion proteins of either the DNA binding domain of GAL4 (DB, pPC86) or the transcription activation domain of GAL4 (TA, pPC97) (Ref. 29 and see "Materials and Methods"). The transformants carrying the two different fusion proteins were first selected by dextrose synthetic drop-out medium with no supplement of leucine and tryptophan (SD, -leu, -trp, +his) to ensure that in different combinations the transformants have both pPC97 and pPC86 plasmids. Identical numbers of cells in each combination were also dotted on the same medium without histidine (SD, -leu, -trp, -his) to test the protein interaction mediated growth. The transformants were allowed to grow at 30 °C for 48 to 72 h. A, interaction of Kvbeta 2 with the NH2-terminal domain of Kv1.4. Four plasmid combinations are listed on the right. The vector construction of the Kv1.4 fragments has been described in Xu et al. (29). Their growth on the SD, -leu, -trp, +his medium is shown in the middle panel on the left. Their growth on the SD, -leu, -trp, -his medium is shown in the lower panel on the left. B, subfamily-specific interaction of Kvbeta 2 with the NH2-terminal domains of K+ channels. The NH2-terminal domains of eight K+ channel alpha -subunits of four subfamilies were subcloned (29) and subjected to the yeast two-hybrid analysis. The subcloned coding sequences are: NShB (aa 1-227), Nshabll (aa 1-435), Nshaw2 (aa 3-174), Nshal2 (aa 38-185), NKv1.4 (aa 1-310), NKv2.1 (aa 1-182), NKv3.1 (aa 1-180), and NKv4.2 (aa 1-183). The eight plasmid combinations are listed on the right. Their growth on the SD, -leu, -trp, +his medium is shown in the middle panel on the left. Their growth on the SD, -leu, -trp, -his medium is shown in the lower panel on the left.
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The ability of Kvbeta 2 to interact with the NH2-terminal domain of Kv1.4 suggests that the resultant association may be essential for Kvbeta 2 to interact with alpha -subunits, as seen in biochemical copurification. In the case of Kvbeta 1, its subfamily-specific association with the NH2-terminal domains of Kv1 alpha -subunits has been shown to be essential for the Kvbeta 1-mediated inactivation (28, 36). Coimmunoprecipitation of K+ channel polypeptides in rat brain has indicated that Kvbeta 2 interacts with Kv1.2 and Kv1.4, but not Kv2.1 (24, 39). To further test the specificity of the Kvbeta 2, pairwise combinations of Kvbeta 2 and the NH2-terminal domains of eight different alpha -subunits were analyzed with the yeast two-hybrid system. The eight alpha -subunits included were: Shaker B (40-42), Shabll, Shaw2, and Shal2 from Drosophila (9); Kv1.4 (or RCK4) (43), Kv2.1 (or DRK1) (44), Kv3.1 (or NGK2b) (45), and Kv4.2 (or rShal1) (46, 47) from rat. These genes belong to the four major subfamilies, one fly gene and one rat gene for each subfamily. Among the selected NH2-terminal domains, Kvbeta 2 interacts only with the NH2-terminal domains of ShB and Kv1.4 (Fig. 3B, numbers 1 and 5), both of which belong to the Kv1 subfamily. Furthermore, the Kvbeta 2 interacting site was mapped to aa 174-306 within the NH2-terminal domain of Kv1.4 (data not shown), which coincides precisely with the domain that interacts with Kvbeta 1 (28, 36). Thus, both Kvbeta 1 and Kvbeta 2 interact subfamily-specifically with the Kv1 alpha -subunits and share the same binding site on the alpha -subunits.


Fig. 3. The beta -beta interaction tested by the yeast two-hybrid system. Experiments similar to that described in Fig. 2 were carried out to test the homomeric and heteromeric interaction between Kvbeta 2 (aa 1-367) and Kvbeta 1 (aa 1-401). Four plasmid combinations are listed on the right (see "Materials and Methods" for vector construction). Their growth on the SD, -leu, -trp, +his medium is shown on the left (middle panel). Their growth on the SD, -leu, -trp, -his medium is shown in the lower panel on the left.
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Formation of Homo- and Heteromultimeric beta -beta Complexes

Based on the hydrodynamic estimates, the alpha -dendrotoxin acceptor (or Kv1.2) complex contains more than one Kvbeta 2 subunit per complex (21). It is not clear, however, whether Kvbeta 2 can form an oligomeric complex in the absence of alpha -subunits. Additionally, since expression patterns of Kvbeta 1 and Kvbeta 2 overlap in vivo (8, 22, 39, 52), it would be interesting to examine whether different beta -subunits can interact with each other to form heteromultimers. Fig. 3 shows that Kvbeta 2 can indeed associate to form homomultimers as the yeast transformants grow in the selection medium lacking histidine (Fig. 3, number 3). The known potassium channels in yeast have distinctive topology and belong to a subclass different from the Shaker-like potassium channels (48). Therefore, the above result supports that Kvbeta 2 is capable of interacting with itself in the absence of alpha -subunits.

Both Kvbeta 1 and Kvbeta 2 are expressed in rat brain with overlapping expression patterns (8, 22, 39). Because Kvbeta 2 forms multimers in the absence of alpha -subunits (Fig. 3, number 3) and has considerable overall sequence homology (73%) to Kvbeta 1, we tested the possible interaction between Kvbeta 1 and Kvbeta 2 and found that Kvbeta 1 and Kvbeta 2 can also interact (Fig. 3, number 4). This implies that Kvbeta 1 and Kvbeta 2 can form heteromultimers in the absence of the pore-forming alpha -subunits.

Inhibition of the Kvbeta 1-mediated Inactivation by Kvbeta 2

Both Kvbeta 2 and Kvbeta 1 interact with the Kv1 alpha -subunits by recognizing the same region in the Kv1 alpha -subunits (Fig. 2 and Ref. 28). Additionally, Kvbeta 2 interacts with itself and/or Kvbeta 1 to form homo- and/or heteromultimers (Fig. 3). Because Kvbeta 1, not Kvbeta 2, induces the fast inactivation of the Kv1 alpha -subunits that lack fast inactivation (Fig. 1), these data suggest that one potential function of Kvbeta 2 would be to alter the efficacy of the Kvbeta 1-mediated inactivation. One predicted outcome would be that Kvbeta 2 weakens the ability of Kvbeta 1 to inactivate, as Kvbeta 2 may compete with Kvbeta 1 for the binding site on alpha -subunits and/or associate with Kvbeta 1 to form Kvbeta 1-Kvbeta 2 heteromultimers containing fewer inactivation particles.

One experiment to test this hypothesis would be to coexpress Kvbeta 1 and a compatible alpha -subunit in the presence or absence of Kvbeta 2 and ask whether Kvbeta 2 alters the ability of Kvbeta 1 to inactivate. We cotransfected COS cells with noninactivating ShBDelta (6-46) and Kvbeta 1 in a 1:6 plasmid ratio of alpha /Kvbeta 1. Fig. 4A shows three representative traces, one from each group, that were superimposed and normalized. These traces were recorded by stepping up the holding potential from -87 mV to a test potential of +13 mV for a duration of 300 ms. ShBDelta (6-46) alone produced a trace with fast activating kinetics lacking N-type fast inactivation. When Kvbeta 1 was included in the transfection, we observed a majority of transfected cells that show the Kvbeta 1-mediated fast inactivation (Fig. 4A). If, however, both Kvbeta 1 and Kvbeta 2 were included in a plasmid ratio of alpha /Kvbeta 1/Kvbeta 2 of 1:6:5, much fewer transfected cells showed fast interaction induced by Kvbeta 1 (see below and Fig. 4B).


Fig. 4. Inhibition of the Kvbeta 1-mediated inactivation by Kvbeta 2. A, normalized K+ currents obtained by whole-cell voltage clamp recording. The current responses were recorded from COS cells transfected with ShBDelta (6-46), ShBDelta (6-46) + Kvbeta 1, or ShBDelta (6-46) + Kvbeta 1 + Kvbeta 2. Plasmid inputs were 3 µg for ShBDelta (6-46), 18 µg for Kvbeta 1, and 15 µg for Kvbeta 2. Typical responses to a voltage step from -87 mV to +13 mV of one cell from each group were normalized according to the peak response and superimposed. The traces for ShBDelta (6-46) + Kvbeta 1 and ShBDelta (6-46) + Kvbeta 1 + Kvbeta 2 have been fit by a two-exponential function to yield inactivation constants. ShBDelta (6-46) + Kvbeta 1: A2/(A1 + A2) = 0.19; tau 1 = 11.9 ms; tau 2 = 154 ms. ShBDelta (6-46) + Kvbeta 1 + Kvbeta 2: A2/(A1 + A2) = 1; tau 1 (not available since A1 = 0); tau 2 = 178 ms. B, distribution of inactivation time constants. A total of 17 cells positive in Shaker currents were recorded for ShBDelta (6-46), 40 cells recorded for the ShBDelta (6-46) + Kvbeta 1 transfection, and 44 cells were recorded for the ShBDelta (6-46) + Kvbeta 1 + Kvbeta 2 transfection. The decay phase of current responses (300 ms duration) to a voltage step from -87 mV to +13 mV was fit by a double exponential function to obtain the onset parameters of inactivation. The cell number (in percentage normalized to the total cell number recorded for that group) was plotted against the inactivation constants. For a given recorded trace, if the response shows both fast and slow inactivation, only tau 1 is used in this plot. If a trace shows no fast inactivation (i.e. A1 = 0), the tau 2 is used in this plot. Top panel, ShBDelta (6-46); middle panel, ShBDelta (6-46) + Kvbeta 1; bottom panel, ShBDelta (6-46) + Kvbeta 1 + Kvbeta 2. The statistical significance was calculated according to a standard formula for comparison of two proportions (see "Materials and Methods"). Comparison of ShBDelta (6-46) + Kvbeta 1 with ShBDelta (6-46) + Kvbeta 1 + Kvbeta 2: z = 4.111 and p < 0.001. C, expression of Kvbeta 1 and Kvbeta 2 polypeptides in COS cells detected by immunoblot analysis. Total protein lysates from the three groups of transfected COS cells (see legend of A) were prepared. The Kvbeta 1 and Kvbeta 2 polypeptides were detected by the 12CA5 monoclonal antibody according to the protocol described in the legend to Fig. 1. Lane 1, COS transfected with ShBDelta (6-46) alone; lane 2, ShBDelta (6-46) + Kvbeta 1; lane 3, ShBDelta (6-46) + Kvbeta 1 + Kvbeta 2. The Kvbeta 1 and Kvbeta 2 signals are marked and labeled on the side.
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The lack of fast inactivation by Kvbeta 1 in the presence of Kvbeta 2 in the example shown in Fig. 4A could have resulted from variable transfection rates of the three plasmids. To address this, we recorded 17 Shaker-positive cells for the ShBDelta (6-46) transfection, 40 cells for the ShBDelta (6-46) + Kvbeta 1 transfection, 44 cells for the ShBDelta (6-46) + Kvbeta 1 + Kvbeta 2 transfection. Since recorded cells were selected solely based on the presence of Shaker current and the expression of CD4 antigen that was cotransfected in all experiments, the percentage of recorded cells in a given transfection shown, fast and/or slow inactivation can then be determined. Among the recorded traces, some exhibited both fast and slow inactivation, the others showed only slow inactivation. The traces were fit by a double exponential function, and the resultant inactivation constants were plotted against the cell number in percentage (Fig. 4B). Fig. 4B shows plots using one inactivation constant per recorded cell, i.e. if the inactivation consists of two (fast and slow) components, only the fast inactivation constant was plotted. For the ShBDelta (6-46) transfection, we observed that all recorded cells lacked the fast inactivation and gave a slow inactivation constant (tau 2) larger than 128 ms (Fig. 4B, top panel). In the 40 recorded cells for ShBDelta (6-46) + Kvbeta 1 (Fig. 4B, middle panel), we observed that more than 60% of recorded cells possessed a fast inactivation component (tau 1 = 1-64 ms), while about 35% of cells showed no fast inactivation, which presumably is due to low or no expression of Kvbeta 1. By contrast, we observed that only 17% of cells showed fast inactivation when Kvbeta 2 was coexpressed (Fig. 4B, bottom panel). As the amount of plasmid input for both ShBDelta (6-46) and Kvbeta 1 was identical in both transfections, it is unlikely that the population of the recorded cells, which showed no fast inactivation, is due to the lack of Kvbeta 1 plasmids. Furthermore, statistical analysis shows that the difference was statistically significant (see legend to Fig. 4B and "Materials and Methods").

Despite the constant plasmid input of Kvbeta 1 in both experiments, the inhibition of the Kvbeta 1-mediated inactivation by Kvbeta 2 can be subjected to several interpretations. For example, it is not known whether the presence of Kvbeta 2 decreases the expression of other subunits at the protein level and/or at the level of channel surface expression, thereby resulting in the inhibition of the Kvbeta 1-mediated inactivation. To examine whether coexpression of beta -subunits differentially altered the channel surface expression, we plotted the current amplitude of ShBDelta (6-46) in the presence of Kvbeta 1 and Kvbeta 1 + Kvbeta 2. The result indicates that the averaged current amplitudes were similar: 6.5 nA for the ShBDelta (6-46) + Kvbeta 1 transfection and 6.7 nA for the ShBDelta (6-46) + Kvbeta 1 + Kvbeta 2 transfection (p > 0.2, Student's t test). Furthermore, there was no obvious correlation between current amplitude and inactivation properties (data not shown). To examine the expression level of Kvbeta 1 in the presence or absence of Kvbeta 2, aliquots of the transfected cells in the experiment (Fig. 4A) were collected, and the total cell lysates were prepared and separated by SDS-polyacrylamide gel electrophoresis. Indeed, the Kvbeta 1 expression was found to be comparable, regardless of the presence of Kvbeta 2 (Fig. 4C, lanes 2 and 3). Together, these results suggest that when both Kvbeta 1 and Kvbeta 2 are expressed in the cells, Kvbeta 2 is capable of inhibiting the Kvbeta 1-mediated inactivation. Because the Kvbeta 1 and ShBDelta (6-46) expression remained relatively constant, both in the presence and absence of Kvbeta 2, the inhibition by Kvbeta 2 is likely to be caused by differential subunit assembly, i.e. Kvbeta 2 competes with Kvbeta 1 for the binding site on alpha -subunits and/or Kvbeta 1 and Kvbeta 2 form heteromultimeric complex(es).

Regions Involved in beta -beta and alpha -beta Interactions

If the differential subunit assembly indeed plays a role in inhibiting the Kvbeta 1-mediated inactivation, then the binding of Kvbeta 2 to alpha -subunits and/or Kvbeta 1 would be essential for the Kvbeta 2-mediated inhibition.

Deletion analysis for Kvbeta 1 has shown that the conserved core region of Kvbeta 1, i.e. amino acids 73-401, is sufficient for the Kvbeta 1-alpha interaction (28). Among the 47 positions that harbor different residues between Kvbeta 1 and Kvbeta 2, 27 are conservative changes. Additionally, both Kvbeta 1 and Kvbeta 2 recognize the same subset of alpha -subunits, i.e. the Kv1 alpha -subunits. These suggest that the corresponding region in Kvbeta 2 may serve a similar function in binding to the alpha -subunits. Indeed, when a truncated fragment representing the conserved core region of Kvbeta 2 was subjected to the yeast two-hybrid test, the core region is sufficient to interact with the NH2-terminal domain of ShB (NShB) (Fig. 5).


Fig. 5. Regions in beta -subunits that are involved in alpha -beta and beta -beta interactions tested by the yeast two-hybrid system. A, deletion mapping of the region(s) in Kvbeta 1 for interaction with the NH2-terminal domain of ShB (NShB) and Kvbeta 2. A diagram representing the coding sequence of Kvbeta 1 is shown. The filled box indicates the COOH-terminal "core region" of Kvbeta 1. Different coding sequences, as indicated in parentheses, were cloned into the pPC97 vector for the yeast two-hybrid test together with either pPC86-NShB or pPC86-Kvbeta 2 as described in the legend to Fig. 2 and under "Materials and Methods." The + indicates growth, the - indicates no growth. B, interaction of Kvbeta 2 and Delta Kvbeta 2(39-367) with NShB and Kvbeta 2, as tested by the yeast two-hybrid system. Data representation is identical to that described in A.
[View Larger Version of this Image (14K GIF file)]

One possibility for inhibiting the Kvbeta 1-mediated inactivation is that Kvbeta 2 can somehow interact with the inactivation particle of Kvbeta 1, which is located at the NH2 terminus (8). This interaction may lead to the defective activity of Kvbeta 1 to inactivate the Kv1 alpha -subunits. Taking this into consideration, we chose to first carry out deletion analysis using Kvbeta 1 to map the region involved in the Kvbeta 1-Kvbeta 2 interaction to test whether the inactivation particle is necessary for the heteromultimeric beta -beta interaction. A total of 11 Kvbeta 1 deletion mutants were constructed (Fig. 5A). Their ability to interact with Kvbeta 2 was tested by the yeast two-hybrid system using growth selection. The minimal region in Kvbeta 1 required for interacting with Kvbeta 2 was mapped to the conserved core region, which indicates that the NH2-terminal 72 residues of Kvbeta 1 are not required for the Kvbeta 1-Kvbeta 2 interaction in the yeast two-hybrid test. Similar to Kvbeta 1, the corresponding core region of Kvbeta 2 is sufficient for interacting with Kvbeta 2 (Fig. 5B). Thus, the conserved core region of beta -subunits is involved in both alpha -beta and beta -beta interactions.

Inhibition of the Kvbeta 1-mediated Inactivation by Truncated Kvbeta 2 Subunits

The results in Fig. 5 suggest that the formation of heteromultimeric alpha -Kvbeta 2 and/or Kvbeta 1-Kvbeta 2 complexes is responsible for Kvbeta 2 to inhibit the Kvbeta 1-mediated inactivation. If this is true, one prediction is that the truncated Kvbeta 2 polypeptide containing the mapped interacting region, i.e. the conserved core region, should be capable of inhibiting the Kvbeta 1-mediated inactivation. To test this, we constructed two Kvbeta 2 deletion mutants: Delta Kvbeta 2(39-367) and Delta Kvbeta 2(39-316) (Fig. 6A). The Delta Kvbeta 2(39-367) mutant contains the intact interacting region mapped by the yeast two-hybrid analysis (Fig. 5), while 51 residues COOH-terminal to the interacting region of Kvbeta 2 were truncated in Delta Kvbeta 2(39-316). The resultant mutant can no longer interact with either Kvbeta 1 or NH2-terminal domains of the Kv1 alpha -subunits (Fig. 5). Because the ability to interact should correlate with the activity in inhibiting the Kvbeta 1-mediated inactivation, we carried out experiments similar to those in Fig. 4, which involved cotransfecting ShBDelta (6-46) and Kvbeta 1 in the presence of either Delta Kvbeta 2(39-367) or Delta Kvbeta 2(39-316), and examined the subsequent inactivation properties. Fig. 6B shows that the protein levels of Kvbeta 2 and the two deletion mutants are comparable. Among the 39 recorded Shaker-positive cells transfected in the presence of Delta Kvbeta 2(39-367), we found only 22.5% of cells that showed fast inactivation (Fig. 6, C and D, panel b). Indeed, Delta Kvbeta 2(39-367) acts similarly to Kvbeta 2 by decreasing the number of cells that exhibit the fast inactivation (Fig. 6, C and D, panels a and b). The difference between Kvbeta 1 alone and Kvbeta 1 + Delta Kvbeta 2(39-367) was statistically significant (p < 0.001 and see legend to Fig. 6D). In contrast, among the 49 recorded cells that were transfected in the presence of Delta Kvbeta 2(39-316), 61% of cells showed fast inactivation. The distribution of inactivation constant from this group of cells is similar to that obtained from cells transfected by ShBDelta (6-46) + Kvbeta 1 (Fig. 6D, panels c and d). Thus, the ability of the mutated Kvbeta 2 to interact with ShBDelta (6-46) and/or Kvbeta 1 directly correlates with their ability to inhibit the Kvbeta 1-mediated inactivation.


Fig. 6. Inhibition of the Kvbeta 1-mediated inactivation by truncated Kvbeta 2 subunits. A, schematic diagram of Kvbeta 2 and its truncations which were subcloned and expressed in COS cells. The filled gray box indicates the COOH-terminal conserved core region of Kvbeta 2. The Delta Kvbeta 2(39-367) mutant is a deletion of Kvbeta 2 that contains only the COOH-terminal core region. The Delta Kvbeta 2(39-316) mutation was constructed by further deleting 51 amino acids from its COOH terminus of the core region. B, immunoblot detection of Kvbeta 1 and Kvbeta 2 mutants expressed in COS cells. COS cells were transfected with ShBDelta (6-46) and Kvbeta 1 in the presence of either Delta Kvbeta 2(39-367) or Delta Kvbeta 2(39-316). Aliquots of the transfected cells were collected and subjected to immunoblot analysis using the mAb12CA5 (see the legend to Fig. 1C for the experimental protocol). The plasmid inputs were 3 µg for ShBDelta (6-46), 18 µg for Kvbeta 1, and 15 µg for either Delta Kvbeta 2(39-367) or Delta Kvbeta 2(39-316). The identity of the mAb12CA5 signals is marked and indicated on the side. C, whole-cell voltage clamp recordings were obtained from transfected COS cells described in B. A total of 39 cells positive for Shaker current were recorded for the ShBDelta (6-46) + Kvbeta 1 + Delta Kvbeta 2(39-367) transfection, 49 cells for the ShBDelta (6-46) + Kvbeta 1 + Delta Kvbeta 2(39-316) transfection. Typical current responses of one cell from each transfection are shown. Voltage steps are from -87 mV to +33 mV in 20-mV increments. D, distribution of inactivation time constants. The recorded traces from the two groups of cells were analyzed according to the procedures described and as in the legend to Fig. 4. Inactivation time constant distribution was plotted against percent of recorded cells. Panel a, ShBDelta (6-46) + Kvbeta 1 + Kvbeta 2; panel b, ShBDelta (6-46) + Kvbeta 1 + Delta Kvbeta 2(39-367); panel c, ShBDelta (6-46) + Kvbeta 1; panel d, ShBDelta (6-46) + Kvbeta 1 + Delta Kvbeta 2(39-316). Statistical analyses of the two-group comparison (see legend to Fig. 4B and "Materials and Methods") were: za,b = 0.555, p = 0.291; za,c = 4.111, p < 0.001; za,d = 4.387, p < 0.001; zc,d = 0.165, p = 0.433.
[View Larger Version of this Image (24K GIF file)]


DISCUSSION

Using transient expression of different combinations of alpha -subunits and beta -subunits, we have observed a functional role of Kvbeta 2 in modulating channel inactivation. Since the activity of Kvbeta 2 to inhibit the Kvbeta 1-mediated inactivation directly correlates with the ability of Kvbeta 2 to associate with alpha -subunits and Kvbeta 1, differential subunit assembly is a likely mechanism responsible for the Kvbeta 2 activity. This subunit interaction may be important for tuning the K+ channel diversity in vivo.

Consistent with the biochemical data from copurification of Kv1.2 and Kvbeta 2 (7, 22), our results (Fig. 2) show that Kvbeta 2 interacts with the NH2-terminal domains of alpha -subunits in a subfamily-specific manner. Curiously, a recent report suggested that both Kvbeta 1 and Kvbeta 2 interact with Kv1 and Kv4 alpha -subunits (23). This is inconsistent with results from biochemical binding and electrophysiological analysis (28, 36). Because Kvbeta 2 plays a role in both channel expression (24) and channel properties (this report), important future experiments would be to investigate the in vivo specificity of alpha -beta interaction.

The formation of heteromultimeric complexes as a mechanism to increase K+ current diversity has been studied in several channel systems. This includes the formation of functional channels by different pore-forming subunits (e.g. nicotinic acetylcholine receptor, voltage-gated K+ channels, etc.) and by assembly of pore-forming subunits with various auxiliary subunits (e.g. voltage-gated sodium or calcium channels) (see review by Catterall (49)). The heteromultimeric oligomerization by auxiliary subunits in the absence of pore-forming subunits has not been reported. The biochemical evidence of two homologous auxiliary subunits "competing" with same set of alpha -subunits and the formation of heteromultimeric auxiliary subunits in the absence of pore-forming subunits suggests yet another potential mechanism to create and tune their electrical diversity. The ability to form homo- or heteromultimers of beta -subunits in the absence of alpha -subunits does not necessarily imply the alpha -beta and beta -beta subunit assembly that happens in separate steps in vivo. Future experiments are needed to determine the physiological existence of heteromeric complexes of beta -subunits and molecular cascade for assembling alpha -beta heteromultimers.

The results reported here demonstrate that Kvbeta 2 is an active player in determining the fast inactivation mediated by other beta -subunits. How does Kvbeta 2 inhibit the Kvbeta 1-mediated inactivation? Our data can be best explained by the following mechanism (Fig. 7A). In the absence of Kvbeta 2, the expression of Kvbeta 1 will permit it to coassemble with compatible alpha -subunits. Depending upon whether the interacting alpha -subunits contain an inactivation gate, Kvbeta 1 either accelerates or induces fast inactivation (Fig. 7A, I). In the presence of the high concentration of Kvbeta 2, Kvbeta 2 occupies most of the sites on alpha -subunits as homomultimeric Kvbeta 2 complexes. As a result, it prevents (or removes) Kvbeta 1 from interacting with alpha -subunits (Fig. 7A, II), thereby inhibiting the Kvbeta 1-mediated inactivation. The coexpression experiments in the present study show that Kvbeta 2 inhibited the Kvbeta 1-mediated inactivation, consistent with the results of immunoblot analyses in which we found that the expression of Kvbeta 2, despite its comparable plasmid input, was considerably higher than that of Kvbeta 1 (Figs. 1C, 4C, and 6B). Thus, under the conditions of our experiments, most alpha -subunits presumably were occupied by homomultimeric Kvbeta 2 complexes, which prevents the limited numbers of Kvbeta 1 subunits to bind, thereby eliminating the Kvbeta 1-mediated inactivation without altering the protein expression of Kvbeta 1 (Fig. 4D).


Fig. 7. A model for the Kvbeta 2-mediated inhibition. A, a schematic diagram illustrates the postulated Kvbeta 2-mediated inhibition. The model may not represent the actual stoichiometry of beta -beta complex(es). B, comparison of the four well characterized beta -subunits.
[View Larger Version of this Image (19K GIF file)]

Conceivably, one should observe an intermediate situation where Kvbeta 1 and Kvbeta 2 are present in a comparable concentration (or different concentrations with an appropriate ratio for heteromultimeric interaction, since the Kvbeta 1 and Kvbeta 2 may have considerable difference in affinity for subunit interaction). Under such conditions, most compatible alpha -subunits should be interacting with heteromultimeric beta -complexes to produce intermediate effects, i.e. Kvbeta 2 weakens but does not remove the Kvbeta 1-mediated inactivation. In an attempt to test this, we performed experiments with lower inputs of Kvbeta 2 plasmid. Thus far, we have not identified an input plasmid ratio that gives rise to currents with a prominent intermediate inactivation constant. A different approach to alter protein expression level for beta -subunits might be necessary to test this hypothesis. The failure to identify intermediate inactivation could also be interpreted by other mechanism(s). For example, compared with Kvbeta 1, Kvbeta 2 has considerably higher affinity for alpha -subunits and/or itself. In this case, the higher affinity and/or avidity for alpha -Kvbeta 2 and/or Kvbeta 2-Kvbeta 2 interactions would yield predominantly alpha -Kvbeta 2 complex(es).

In mammalian systems, two genes encoding three forms of mammalian beta -subunits, i.e. Kvbeta 1, Kvbeta 3 (a putative splice variant of Kvbeta 1), and Kvbeta 2, have been found expressed in brain with an overlapping expression pattern (7, 8, 39). Although Kvbeta 3 has not been demonstrated directly to bind either alpha -subunits or beta -subunits, the fact that the Kvbeta 3 amino acid sequence within the interacting region is identical to that of Kvbeta 1 suggests that Kvbeta 3 is likely to share features for subunit interaction found in Kvbeta 1 and Kvbeta 2 (17, 18, 20). Based on the results of studying subunit interaction between alpha -subunits and beta -subunits, an interesting new regulatory pathway is emerging. All cloned beta -subunits consist of a conserved core region critical for alpha -beta and beta -beta interaction and variable NH2-terminal domains that determine the modulatory activity (Fig. 7B). Their distinct modulatory effects on alpha -subunits and ability to form heteromultimers have revealed yet another potential in vivo mechanism for creating and tuning the diversity of potassium currents. Although our present study probes the formation of heteromultimers by examining the protein-protein interaction and the resultant alterations of inactivation properties, there is increasing evidence suggesting that the functional roles of beta -subunits may not be restricted to modulation of inactivation (16, 24, 50). Thus, future experiments should be focused on addressing whether the heteromultimeric assembly of beta -subunits is indeed present in vivo and whether these heteromultimeric alpha -beta combinations could specify a wide spectrum of modulatory activities, which may include, but are not limited to, tuning inactivation properties and surface expression of alpha -subunits in vivo.


FOOTNOTES

*   This work was supported in part by grants from the National Institutes of Health, the Council for Tobacco Research, Inc., and by an American Heart Association-Pfizer award (to M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Neuroscience fellow of the Alfred P. Sloan Foundation and the Esther A. and Joseph Klingenstein Fund. To whom correspondence should be addressed: Dept. of Physiology, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., WBSB 216, Baltimore, MD 21205. Tel.: 410-614-3692; Fax: 410-614-1001; E-mail: min_li{at}qmail.bs.jhu.edu.
1   M. Bezanilla, J. Xu, and M. Li, unpublished results.
2   The abbreviation used is: aa, amino acids.

ACKNOWLEDGEMENTS

We thank Magdalena Bezanilla for the Delta Kvbeta 2(39-316) construct; Dr. Weifeng Yu for help on patch clamp techniques; Dr. Lily Jan for ShB and rshal1 (Kv4.2); Dr. R. Joho and Dr. A Brown for DRK1; Dr. D. Mckinnon for RCK1; Dr. L. Salkoff for fshal2, fshaw2, and fshabl1; and Dr. R. Aldrich for the ShBDelta (6-46) mutant. We also thank Magdalena Bezanilla, Gerda Breitwieser, Xiaodong Li, Rajini Rao, and Weifeng Yu for critical reading of this manuscript.


REFERENCES

  1. Connor, J. A., and Stevens, C. F. (1971) J. Physiol. (Lond.) 213, 21-30 [Medline] [Order article via Infotrieve]
  2. Byrne, J. H. (1980) J. Neurophysiol. 43, 651-668 [Free Full Text]
  3. Rogawski, M. A. (1985) Trends Neurosci. 8, 214-219 [CrossRef]
  4. Hille, B. (1991) Ionic Channels of Excitable Membrane, pp. 58-75 and 99-116, Sinauer, Sunderland, MA
  5. Jan, L. Y., and Jan, Y. N. (1990) Trends Neurosci. 13, 415-419 [CrossRef][Medline] [Order article via Infotrieve]
  6. Salkoff, L., Baker, K., Butler, A., Covarrubias, M., Pak, M. D., and Wei, A. (1992) Trends Neurosci. 15, 161-166 [CrossRef][Medline] [Order article via Infotrieve]
  7. Scott, V. E., Rettig, J., Parcej, D. N., Keen, J. N., Findlay, J. B., Pongs, O., and Dolly, J. O. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1637-1641 [Abstract]
  8. Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej, D. N., Dolly, J. O., and Pongs, O. (1994) Nature 369, 289-294 [CrossRef][Medline] [Order article via Infotrieve]
  9. Butler, A., Wei, A., and Salkoff, L. (1990) Nucleic Acids Res. 18, 2173-2174 [Medline] [Order article via Infotrieve]
  10. Wei, A., Covarrubias, M., Butler, A., Baker, K., Pak, M., and Salkoff, L. (1990) Science 248, 599-603 [Medline] [Order article via Infotrieve]
  11. Chandy, K. G., and Gutman, G. A. (1993) Trends Pharmacol. Sci. 14, 434 [Medline] [Order article via Infotrieve]
  12. Zhao, B., Rassendren, F., Kaang, B. K., Furukawa, Y., Kubo, T., and Kandel, E. R. (1994) Neuron 13, 1205-1213 [Medline] [Order article via Infotrieve]
  13. Jegla, T., and Salkoff, L. (1995) Recept. Channels 3, 51-60 [Medline] [Order article via Infotrieve]
  14. MacKinnon, R. (1991) Nature 350, 232-235 [CrossRef][Medline] [Order article via Infotrieve]
  15. Li, M., Unwin, N., Stauffer, K. A., Jan, Y. N., and Jan, L. Y. (1994) Curr. Biol. 4(2), 110-115
  16. Chouinard, S. W., Wilson, G. F., Schlimgen, A. K., and Ganetzky, B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6763-6767 [Abstract]
  17. England, S. K., Uebele, V. N., Shear, H., Kodali, J., Bennett, P. B., and Tamkun, M. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6309-6313 [Abstract]
  18. Majumder, K., De Biasi, M., Wang, Z., and Wible, B. A. (1995) FEBS Lett. 361, 13-16 [CrossRef][Medline] [Order article via Infotrieve]
  19. McCormack, K., McCormack, T., Tanouye, M., Rudy, B., and Stuhmer, W. (1995) FEBS Lett. 370, 32-36 [CrossRef][Medline] [Order article via Infotrieve]
  20. Morales, M. J., Castellino, R. C., Crews, A. L., Rasmusson, R. L., and Strauss, H. C. (1995) J. Biol. Chem. 270, 6272-6277 [Abstract/Free Full Text]
  21. Parcej, D. N., Scott, V. E., and Dolly, J. O. (1992) Biochemistry 31, 11084-11088 [Medline] [Order article via Infotrieve]
  22. Scott, V. E. S., Parcej, D. N., Keen, J. N., Findlay, J. B. C., and Dolly, J. O. (1990) J. Biol. Chem. 265, 20094-20097 [Abstract/Free Full Text]
  23. Nakahira, K., Shi, G., Rhodes, K. J., and Trimmer, J. S. (1996) J. Biol. Chem. 271, 7084-7089 [Abstract/Free Full Text]
  24. Shi, G., Nakahira, K., Hammond, S., Rhodes, K., Schechter, L., and Trimmer, J. (1996) Neuron 16, 843-852 [Medline] [Order article via Infotrieve]
  25. Feilotter, H. E., Hannon, G. J., Ruddell, C. J., and Beach, D. (1994) Nucleic Acids Res. 22, 1502-1503 [Medline] [Order article via Infotrieve]
  26. Sambrook, J., Fristsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  27. Li, M., Jan, Y. N., and Jan, L. Y. (1992) Science 257, 1225-1230 [Medline] [Order article via Infotrieve]
  28. Yu, W. F., Xu, J., and Li, M. (1996) Neuron 16, 441-453 [Medline] [Order article via Infotrieve]
  29. Xu, J., Yu, W., Jan, Y. N., Jan, L. Y., and Li, M. (1995) J. Biol. Chem. 270, 24761-24768 [Abstract/Free Full Text]
  30. Fields, S., and Song, O. (1989) Nature 340, 245-246 [CrossRef][Medline] [Order article via Infotrieve]
  31. Chevray, P. M., and Nathans, D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5789-5793 [Abstract]
  32. Gietz, D., St, J. A., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids Res. 20, 1425 [Medline] [Order article via Infotrieve]
  33. Hamill, O. P., Marty, A., Nehre, E., Sakmann, B., and Sigworth, F. J. (1981) Pfluegers Arch. 391, 85-100 [Medline] [Order article via Infotrieve]
  34. Barry, P. H. (1994) J. Neurosci. Methods 51, 107-116 [CrossRef][Medline] [Order article via Infotrieve]
  35. Pagano, M., and Gauvreau, K. (1993) Principles of Biostatistics, Duxbury Press, Belmont, CA
  36. Sewing, S., Roeper, J., and Pongs, O. (1996) Neuron 16, 455-463 [Medline] [Order article via Infotrieve]
  37. Hoshi, T., Zagotta, W. N., and Aldrich, R. W. (1990) Science 250, 533-538 [Medline] [Order article via Infotrieve]
  38. Zagotta, W. N., Hoshi, T., and Aldrich, R. W. (1990) Science 250, 568-571 [Medline] [Order article via Infotrieve]
  39. Rhodes, K. J., Keilbaugh, S. A., Barrezueta, N. X., Lopez, K. L., and Trimmer, J. S. (1995) J. Neurosci. 15, 5360-5371 [Abstract]
  40. Kamb, A., Iverson, L. E., and Tanouye, M. A. (1987) Cell 50, 405-413 [Medline] [Order article via Infotrieve]
  41. Tempel, B. L., Papazian, D. M., Schwarz, T. L., Jan, Y. N., and Jan, L. Y. (1987) Science 237, 770-775 [Medline] [Order article via Infotrieve]
  42. Pongs, O., Kecskemethy, N., Muller, R., Krah-Jentgens, I., Baumann, A., Kiltz, H. H., Canal, I., Llamazares, S., and Ferrus, A. (1988) EMBO J. 7, 1087-1096 [Abstract]
  43. Stuhmer, W., Ruppersberg, J. P., Schroter, K. H., Sakmann, B., Stocker, M., Giese, K. P., Perschke, A., Baumann, A., and Pongs, O. (1989) EMBO J. 8, 3235-3244 [Abstract]
  44. Frech, G. C., VanDongen, A. M., Schuster, G., Brown, A. M., and Joho, R. H. (1989) Nature 340, 642-645 [CrossRef][Medline] [Order article via Infotrieve]
  45. Yokoyama, S., Imoto, K., Kawamura, T., Higashida, H., Iwabe, N., Miyata, T., and Numa, S. (1989) FEBS Lett. 259, 37-42 [CrossRef][Medline] [Order article via Infotrieve]
  46. Baldwin, T. J., Tsaur, M. L., Lopez, G. A., Jan, Y. N., and Jan, L. Y. (1991) Neuron 7, 471-483 [Medline] [Order article via Infotrieve]
  47. Roberds, S. L., and Tamkun, M. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1798-1802 [Abstract]
  48. Ketchum, K. A., Joiner, W. J., Sellers, A. J., Kaczmarek, L. K., and Goldstein, S. A. N. (1995) Nature 376, 690-695 [CrossRef][Medline] [Order article via Infotrieve]
  49. Catterall, W. A. (1988) Science 242, 50-61 [Medline] [Order article via Infotrieve]
  50. Castellino, R. C., Morales, M. J., Strauss, H. C., and Rasmusson, R. L. (1995) Am. J. Physiol. 269, H385-H391 [Abstract/Free Full Text]
  51. Li, X., Xu, J., and Li, M. (1997) J. Biol. Chem. 272, 705-708 [Abstract/Free Full Text]
  52. Rhodes, K. J., Monaghan, M. M., Barrezueta, N. X., Nawoschik, S., Bekele-Akcuri, Z., Matos, M. F., Nakahira, K., Schechter, L. E., and Trimmer, J. S. (1996) J. Neurosci. 16, 4846-4860 [Abstract/Free Full Text]

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