Mutations in the Kvbeta 2 Binding Site for NADPH and Their Effects on Kv1.4*

Ravikumar PeriDagger §, Barbara A. Wible, and Arthur M. BrownDagger ||

From the Dagger  Rammelkamp Center for Education and Research, MetroHealth Campus and the  Departments of Physiology and Biochemistry, Case Western Reserve University, Cleveland, Ohio 44109

Received for publication, September 14, 2000, and in revised form, October 2, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Kvbeta 2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kvbeta 2 binds NADP+, and it has been suggested that Kvbeta 2 is an oxidoreductase enzyme (1). To investigate how this function might relate to channel modulation, we made point mutations in Kvbeta 2 in either the NADPH docking or putative catalytic sites. Using the yeast two-hybrid system, we found that these mutations did not disrupt the interaction of Kvbeta 2 with Kvalpha 1 channels. To characterize the Kvbeta 2 mutants functionally, we coinjected wild-type or mutant Kvbeta 2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kvbeta 2 increased both the amplitude and rate of inactivation of Kv1.4 currents. The cellular content of Kv1.4 protein was unchanged on Western blot, but the amount in the plasmalemma was increased. Mutations in either the orientation or putative catalytic sites for NADPH abolished the expression-enhancing effect on Kv1.4 current. Western blots showed that both types of mutation reduced Kv1.4 protein. Like the wild-type Kvbeta 2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kvbeta 2 subunits with Kv1.4. Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kvbeta 2 on Kv1.4 currents from its effect on the rate of inactivation. These results suggest that the binding of NADPH and the putative oxidoreductase activity of Kvbeta 2 may play a role in the processing of Kv1.4.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Voltage-gated K+ (Kv)1 channels are integral membrane proteins that regulate the electrical properties of excitable cells. Channels in the Kv1 subfamily consist of hetero-oligomeric protein complexes comprising four pore-forming alpha  subunits in permanent association with four modulatory beta  subunits (1). Three different Kvbeta subunit genes (beta 1, beta 2, and beta 3) have been cloned from different tissues in several species (2-6). Kvbeta subunits coassemble with Kvalpha subunits in the endoplasmic reticulum and enhance biosynthesis, maturation, and surface expression of voltage-gated K+ channel complexes. Kvbeta 2 increases Kv1.2 (7) and Kv1.4 (8) currents and also increases the cell surface dendrotoxin binding site of Kv1.2 (9). The N terminus of Kvbeta 1.x (beta 1.1, beta 1.2, and beta 1.3) acts as a ball peptide and rapidly inactivates open channels. This fast inactivation resembles the N-type inactivation produced by the N-terminal ball peptide of Kvalpha subunits. The Kvbeta 2 subunit while lacking the inactivation peptide accelerates the N-type inactivation of Kv1.4 and in addition increases Kv1.4 expression (8).

The conserved C-terminal core region of Kvbeta subunits shares a remarkable structural homology to aldo-keto reductases (10). The crystal structure of the conserved core of Kvbeta 2 subunit depicts a 4-fold symmetrical TIM barrel structure with the bound cofactor NADP+ (1). The structure shows that residues Ser-188 and Arg-189 are important for the orientation of the nicotinamide ring of NADP+ and that Asp-85 and Tyr-90 are the putative catalytic site residues of Kvbeta 2. The phenolic moiety of Tyr-90 positioned near the C-4 of nicotinamide ring would be a proton donor for reduction of a putative substrate aldehyde or ketone. The Asp-85 residue is involved in extensive hydrogen bonding and together with Tyr-90 positions the catalytic site as for other aldo-keto reductases. Despite this structural information, it is not known whether Kvbeta subunits function as oxidoreductases and, if so, what their physiological substrates may be.

In this study, we investigated the contribution of these residues to Kvbeta 2 function. We made site-directed point mutations in both the NADPH orientation and putative catalytic sites. The putative catalytic mutant (D85A, Y90F) is referred to as Kvbeta 2 Mut1 and the orientation mutant (S188A, R189L) is referred to as Kvbeta 2 Mut2. We tested the effects of these mutants on both the inactivation properties and the expression levels of Kv1.4 currents in Xenopus oocytes. We report that wild-type Kvbeta 2 increased Kv1.4 expression by enhancing its trafficking to the cell surface. Mutations at either site eliminated the ability of Kvbeta 2 to enhance Kv1.4 expression. However, mutant Kvbeta 2 subunits still interacted with Kv1.4 to increase the rate of inactivation. Thus, the dual effects of Kvbeta 2 regulation of Kv1.4 were shown to be independent by mutating residues involved in NADP+ binding.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Site-directed Mutagenesis-- Site-directed mutations (D85A, Y90F mutant 1; S188A, R189L mutant 2) were made in Kvbeta 2 cDNA using the overlapping polymerase chain reaction method, from which the products were subcloned into the TOPO shuttle vector (Invitrogen) for sequencing. Wild-type and mutant Kvbeta 2 cDNAs were subcloned into the pCR3 vector (Invitrogen), which we modified to contain a poly(A)+ tail and a unique NruI site for linearizing the constructs prior to cRNA synthesis.

Analysis of Protein-Protein Interaction by the Yeast Two-hybrid System-- Protein-protein interactions were monitored with the yeast Matchmaker two-hybrid system (CLONTECH). Kvbeta 2 wild-type and Kvbeta 2 mutant cDNAs were subcloned into the yeast shuttle vector, pGBT9 (DNA binding domain vector). The N terminus of Kv1.4 (amino acids 1-305) was fused to the activation domain vector, pGAD424. Protein-protein interactions were tested in the yeast strain Y190 by cotransformation with pairs of pGBT9 and pGAD424 constructs as described previously (11). Cotransformants were selected on medium lacking tryptophan (trp-) and leucine (leu-) after growth for 2-3 days at 30 °C. Representative colonies were replated on trp-/leu- medium to allow for direct comparison of individual colonies. Transcription of the reporter gene, lacZ, was tested by a beta -galactosidase filter assay.

In Vitro Transcription of RNA and Expression in Xenopus Oocytes-- cRNA was prepared using the T7 mMESSAGE mMACHINE kit (Ambion), and the concentrations were estimated on denaturing agarose gels stained with ethidium bromide by comparison with an RNA mass ladder. Xenopus oocytes were injected with 46 nl of cRNA solution. Final concentrations of cRNA were 2 ng/µl Kv1.4 and 50 ng/µl Kvbeta 2 or Kvbeta 2 mutants.

Electrophysiology-- Whole cell current was measured at room temperature (20-22 °C) using the standard two-electrode voltage clamp technique. Electrodes were filled with 3 M KCl and had a resistance of 0.2-0.5 megohms when immersed in bath solution containing (in mM): 50 KOH, 55 NaOH, 0.5 CaCl2·2H2O, 100 methanesulfonic acid, 2 MgCl2·6H2O, and 10 HEPES, pH 7.3. All chemicals were purchased from Sigma. We used 50 mM K+ in bath solution to slow C-type inactivation and reduce its contribution to the N-type inactivation of Kv1.4. All currents were measured 6 days after injection. Data acquisition and analysis were performed using pCLAMP software (Axon Instruments). Data were low pass-filtered at 2 kHz before digitization at 10 kHz. Data are reported as means ± S.E.

Xenopus Oocyte Fractionation and Western Blotting-- Plasma membranes were prepared from Xenopus oocytes using a previously published procedure (12). Oocytes were homogenized (20 µl/oocyte) in buffer containing 0.25 mM sucrose, 10 mM HEPES 1 mM EGTA, 2 mM MgCl2·6H2O plus 1 µg/ml phenylmethylsulfonyl fluoride with five strokes in a Dounce homogenizer using a loose-fitting pestle. Sheets of plasma membrane were allowed to settle by gravity for 15 min and then washed and resedimented three times. Washed plasma membranes were solubilized in a buffer containing 1% Triton X-100 prior to determination of protein concentration. The supernatant from the initial homogenization was spun at 3,000 × g for 10 min to remove debris, and the internal membranes were pelleted by centrifugation at 50,000 × g for 60 min. Protein concentrations were estimated by the BCA method. For Western blotting, proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted to polyvinylidene difluoride membranes, and blocked with 5% nonfat dry milk in phosphate-buffered saline plus 0.1% Tween 20. Blots were incubated with primary antibodies (monoclonal anti-Kv1.4, Upstate Biotechnology, Inc., 1:500; or polyclonal anti-Kvbeta 2, QCB, 1:5000) for 1 h at room temperature, washed three times (10 min/wash) with phosphate-buffered saline, 0.1% Tween 20, and incubated with secondary antibody (anti-mouse or anti-rabbit horseradish peroxidase conjugate, Amersham Pharmacia Biotech, 1:3000) for 1 h. After three washes in phosphate-buffered saline plus 0.1% Tween 20, the blots were developed with the ECL-Plus detection system (Amersham Pharmacia Biotech). Total membranes were prepared from oocytes by methods previously established in our laboratory (13). The oocytes were ground by 20 strokes in a Dounce homogenizer and spun at 3,000 × g for 10 min to remove debris, and the total membranes were pelleted by centrifugation at 50,000 × g for 60 min.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interaction of Kv1.4 N Terminus with Kvbeta 2 Mutants-- We first tested whether the mutations in the NADPH orientation and NADPH oxidoreductase catalytic sites affected interaction with Kv1.4 channels. Using the yeast two-hybrid assay, we found that neither set of mutations diminished the binding of Kvbeta 2 with the Kv1.4 N terminus, as evidenced by activation of the lacZ reporter gene (Fig. 1).



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Fig. 1.   Yeast two-hybrid interaction of Kvbeta 2 mutants with Kv1.4N. The yeast strain Y190 was cotransformed with the GAL4 binding domain (BD, pGBT9) and activation domain (AD, pGAD424) fusion plasmids as indicated and plated on medium lacking trp and leu. All cotransformants grew on trp-/leu- medium and were tested for activation of the reporter gene, lacZ, by a filter assay. The control reaction did not result in activation of lacZ, but Kvbeta 2 as well as both Kvbeta 2 mutants interacted with Kv1.4N as seen by the positive beta -galactosidase assay.

Effect of Kvbeta 2 Mutants on Kv1.4 Currents-- Kvbeta 2 has been reported to increase both the amplitude and the rate of inactivation of Kv1.4 currents. We investigated the effects of Kvbeta 2 mutations on Kv1.4 currents by coexpressing cRNAs encoding Kv1.4 with wild-type or mutant Kvbeta 2 in Xenopus oocytes. Six days after injection, we observed that Kvbeta 2 increased Kv1.4 current amplitude by 1.6-fold at +80 mV (compare Fig. 2, panels A and B). The current amplitude measured at +80 mV was 15.35 ± 0.78 µA (n = 9) (Fig. 2B) in oocytes expressing a combination of Kv1.4 and Kvbeta 2 compared with 9.55 ± 0.88 µA (n = 7) for oocytes expressing Kv1.4 alone (Fig. 2A). The increase in current was manifest at all potentials above threshold (Fig. 3A). When Kvbeta 2 mutants were examined, the expression-enhancing effect of Kvbeta 2 on Kv1.4 was abolished. In fact, coexpression with Kvbeta 2 mutants produced a decrease in the amplitude of Kv1.4 current, and the decrease occurred at all potentials above threshold (Fig. 3A). At +80 mV, mutations in the NADPH oxidoreductase catalytic site (D85A, Y90F) decreased currents by 63.3% to 4.46 ± 0.24 µA (n = 5) (Fig. 2C), whereas mutations in the NADPH docking site (S188A, R189L) reduced currents by 36.6% to 6.05 ± 0.45 µA (n = 6) (Fig. 2D). The double mutant in which all four residues were simultaneously disrupted also showed a reduction in current amplitude to 7.24 ± 0.36 µA (n = 6), a decrease of 24% (data not shown). The differences in mean current amplitude for Kv1.4 compared with mean current amplitude for Kv1.4 in combination with either Kvbeta 2 or Kvbeta 2 mutants (p < 0.01, one-way analysis of variance) were statistically significant.



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Fig. 2.   Mutations in NADPH docking and oxidoreductase catalytic sites abolish the expression enhancing effects of Kvbeta 2. In Xenopus oocytes, whole cell currents were recorded 6 days post-injection. Currents were evoked from a holding potential of -80 mV by 125-ms depolarizing step pulses to +80 mV in 10 mV increments (shown in insert). The oocytes were either injected with Kv1.4 (2 ng/µl) alone (A) or coinjected with Kvbeta 2 (50 ng/µl) (B), Kvbeta 2 mut1 (50 ng/µl) (C), or Kvbeta 2 mut2 (50 ng/µl) (D). E, peak current amplitudes at +80 mV are depicted in the bar diagram from the current traces shown in A-D. F, normalized current traces at +80 mV depict enhanced N-type inactivation in the presence of Kvbeta 2 and Kvbeta 2 mutants, indicating their interaction with Kv1.4.



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Fig. 3.   Mutations in NADPH docking and oxidoreductase catalytic sites do not significantly alter the biophysical properties of Kvbeta 2. A, current-voltage relationship of currents recorded from Xenopus oocytes. Whole cell currents were evoked from a holding potential of -80 mV by 125-ms depolarizing step pulses to +80 mV in 10 mV increments (current traces shown in Fig.2, A-D). B, coexpression of Kv1.4 with Kvbeta 2 or Kvbeta 2 mutants did not alter the voltage dependence of activation. I/Imax ratios were calculated by dividing the peak current at each potential with the maximal current at +80 mV and plotted as a function of potential. C, steady state inactivation levels of Kv1.4 in the presence and absence of Kvbeta 2 and Kvbeta 2 mutants were similar. Oocytes were held at -90 mV and pulsed to -10 mV in 10 mV increments for 1 s followed by a 450-ms test pulse to +70 mV and repolarization to -90 mV for 6 s (n = 4). Steady state inactivation curves were generated by normalizing the currents at each potential to the maximal currents at +70 mV. The data were fit to the Boltzman equation I/Imax = 1/[1 + exp(V0.5 - V)/k)], where V0.5 is the potential for half-inactivation and k is the slope factor. D, plot of inactivation time constants versus test potential. Inactivating current traces of Kv1.4 in the presence of Kvbeta 2 and Kvbeta 2 mutants were fitted with a single exponential function. The time constants were plotted as a function of potential (n = 4). Inactivation time constants are significantly smaller when Kvbeta 2 or wild-type Kvbeta 2 mutants are coexpressed with Kv1.4, indicating enhanced inactivation; however, they are not significantly different from each other. E, plot of recovery from inactivation. From a holding potential of -90 mV, the oocyte was depolarized to +70 mV for 1 s and subsequently repolarized to -90 mV for time intervals ranging from 100 ms to 2.4 s. This was followed by a 400-ms test pulse to +70 mV (n = 4). The recovery curve is the plot of the ratio of inactivated current during the second pulse, normalized to the amount of inactivating current during the first pulse versus the inter-pulse interval. Kvbeta 2 slowed the recovery from inactivation compared with Kv1.4 alone, whereas the Kvbeta 2 mutants produced an even slower recovery from inactivation.

Yeast two-hybrid data indicated that the mutant Kvbeta subunits were still able to interact with the Kv1.4 N terminus (Fig. 1). As a further test of association, we examined Kv1.4 currents for evidence of increased inactivation produced by association with Kvbeta 2 subunits. In an earlier study (8), we had shown that Kvbeta 2, although absent the inactivation peptide of Kvbeta 1.x, increased the rate of N-type inactivation of Kv1.4. Normalized current traces of Kv1.4 when coexpressed with either Kvbeta 2 or Kvbeta 2 mutants showed that wild-type and mutant Kvbeta 2 enhanced the intrinsic inactivation of Kv1.4 currents to a similar extent (Fig. 2F). Plots of inactivation time constants as a function of membrane potential were also similar (Fig. 3D). At +80 mV, the mean inactivation time constants for Kv1.4, Kv1.4+ beta 2, Kv1.4 + beta 2 mut1, and Kv1.4 + beta 2 mut2 were 49.55 ± 2.3, 25.52 ± 0.37, 21.67 ± 0.18, and 22.32 ± 0.28, respectively. Neither wild-type nor mutant Kvbeta 2 subunits altered the voltage dependence of activation (Fig. 3B) or steady state inactivation (Fig. 3C) of Kv1.4. Recovery from steady state inactivation, which was decreased when Kvbeta 2 was coexpressed with Kv1.4, was slowed further in the presence of the Kvbeta 2 mutants (Fig. 3E). At -90mV, the mean time for half-recovery from inactivation for Kv1.4, Kv1.4 beta 2, Kv1.4 + beta 2 mut1, and Kv1.4 + beta 2 mut2 were 3.35, 3.42, 4.23, and 5.82 s, respectively. To eliminate the possibility of nonspecific effects of Kvbeta 2 mutants on Kv1.4 in oocytes, Kvbeta 2 wild-type and mutants were also coexpressed with Kv2.1, a Kvalpha subunit with which they do not interact. Neither Kvbeta 2 nor the Kvbeta 2 mutants had any effect on Kv2.1 current amplitude or kinetics (data not shown).

Kvbeta 2 Enhances Trafficking of Kv1.4 to the Plasma Membrane-- Our electrophysiological data showed that the expression-enhancing effect of Kvbeta 2 on Kv1.4 was distinct from its effects on the biophysical properties of the channel. The enhancement of Kv1.4 expression by Kvbeta 2 could be caused by: 1) an increase in total Kv1.4 protein, 2) an increase in the number of functional channels at the cell surface without an increase in total Kv1.4 levels, or 3) an increase in opening probability and/or single channel conductance. The third possibility was considered unlikely because the conductance-voltage relationship of Kv1.4 was unaltered by wild-type and mutant Kvbeta 2s (Fig. 3B). To determine whether coexpression of Kvbeta 2 increased total Kv1.4 protein levels, we used Western blotting to examine membrane-enriched fractions from oocytes. Multiple bands of Kv1.4 were detected in oocytes injected with Kv1.4 cRNA: an immature or core-glycosylated ~78-kDa band; and a mature, glycosylated, 105-kDa band, which resolved as a doublet in some experiments (Fig. 4). Coexpression with Kvbeta 2 did not alter the amounts of Kv1.4 protein in the total membrane fraction. However, coexpression with either of the Kvbeta 2 mutants did show a reduction in total Kv1.4 protein.



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Fig. 4.   Kvbeta 2 coexpression did not alter the total membrane associated Kv1.4 protein in Xenopus oocytes, whereas Kvbeta 2 mutations in the NADPH docking and putative oxidoreductase catalytic sites decreased Kv1.4 protein levels. Total membranes were isolated 6 days post-injection from Xenopus oocytes either injected with Kv1.4 (2 ng/µl) alone or coinjected with Kvbeta 2 or Kvbeta 2 mut1 or Kvbeta 2 mut2 (all at 50 ng/µl). Proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted to polyvinylidene difluoride membranes, and probed with either polyclonal anti-Kvbeta 2 (1:5000, QCB; panel A) or monoclonal anti-Kv1.4 (1:500, Upstate Biotechnology, Inc.; panel B). Immunoreactive bands were visualized with ECL-Plus (Amersham Pharmacia Biotech). Kv1.4 appeared as three bands, a 78-kDa core-glycosylated band and a doublet at 105 kDa representing mature-glycosylated bands). Coexpression with wild-type Kvbeta 2 did not increase the total Kv1.4 expression, whereas both of the Kvbeta 2 mutants decreased total Kv1.4 protein levels. Kvbeta 2 blot shows that the mutation in the catalytic site led to decreased beta 2 protein synthesis.

Because Kvbeta 2 did not increase total Kv1.4 protein levels, we explored the possibility that Kvbeta 2 increased currents by enhancing trafficking of Kv1.4 channels to the plasma membrane by physically separating oocyte plasma membranes from internal membranes (i.e. endoplasmic reticulum and Golgi). Western blot analysis of Kv1.4 in these fractions showed an enhanced expression in the plasma membrane when coexpressed with Kvbeta 2 (Fig. 5, right). As expected, only the mature, fully glycosylated ~105-kDa band (which was not resolved into a clear doublet in this gel) was detected in the plasma membrane. In the internal membrane fraction, we found both the immature and mature Kv1.4 bands with no apparent change in expression in the presence or absence of Kvbeta 2 (Fig. 5, left). Consistent with decreased currents, coexpression with Kvbeta 2 mutants showed a reduction of Kv1.4 in both the plasma and internal membrane fractions (Fig. 5). Both wild-type and mutant Kvbeta 2 subunits were detected in both the internal and plasma membrane fractions, providing further evidence for the interaction of mutant Kvbeta 2 subunits with Kv1.4. Although equal amounts of cRNAs were injected for each Kvbeta cDNA, we consistently observed a lower level of expression of Kvbeta 2 mutant 1. It is not known whether this finding reflects an increased degradation of mutant protein, decreased synthesis, or a combination of both.



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Fig. 5.   Kvbeta 2 enhanced the surface expression of Kv1.4, whereas Kvbeta 2 mutations in the NADPH docking and oxidoreductase catalytic sites decreased Kv1.4 protein levels. Plasma membrane and internal membrane fractions were isolated 6 days post-injection from Xenopus oocytes injected with Kv1.4 (2 ng/µl) alone or coinjected with Kvbeta 2, Kvbeta 2 mut1, or Kvbeta 2 mut2 (all at 50 ng/µl). Proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted to polyvinylidene difluoride membranes, and probed with either with monoclonal anti-Kv1.4 (1:500, Upstate Biotechnology, Inc; panels A and B) or polyclonal anti-Kvbeta 2 (1:5000, QCB; panels C and D). Immunoreactive bands were visualized with ECL-Plus (Amersham Pharmacia Biotech). Kv1.4 appeared as two bands in the internal membrane fraction (A), a 78-kDa core-glycosylated band and a 105-kDa mature-glycosylated band. Only the mature, glycosylated, 105-kDa band was seen in the plasma membrane fraction (B). Note that coexpression with wild-type Kvbeta 2 increased Kv1.4 expression in the plasma membrane fraction, whereas both of the Kvbeta 2 mutants decreased Kv1.4 protein levels in the internal as well as the plasma membrane fractions.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Kvbeta 2 increased Kv1.4 current and its rate of inactivation (8). From the present results it appears that the increased Kv1.4 current may be due to a redistribution of Kv1.4 protein to the plasmalemma rather than an increase in the total amount of membrane-associated Kv1.4 protein. Mutating wild-type Kvbeta 2 at the orientation site for NADPH (Mut2) or the putative catalytic site for aldo-keto reductase activity (Mut1) abolished the ability of Kvbeta 2 to increase Kv1.4 current without changing the ability of Kvbeta 2 to increase inactivation. The latter result, together with the persistent yeast two-hybrid interactions, showed that the mutant Kvbeta 2 subunits were still permanently associated with Kv1.4 subunits. However, the Kvbeta 2 mutants produced a reduction in both the plasmalemmal and total membrane-associated Kv1.4 protein, which probably explains the reduction in Kv1.4 currents associated with their coexpression (Fig. 2E).

Our results do not show that NADPH binding by Kvbeta 2 has been abolished, nor do they show whether the effects of the mutations are specific for orientation of the nicotinamide ring or the putative catalytic site for aldo-keto reductase activity. Such studies are possible now that the crystal structure of Kvbeta 2 (1) and the assembly of Kvbeta 1.x with the T1 domain of Kv1.x channels (14) have been resolved. Keeping these assumptions in mind, it may be possible that the aldo-keto reductase activity of Kvbeta 2 is important for the processing and trafficking of Kv1.4


    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL55404 and HL36930 (to A. M. B.) and HL60759 (to B. A. W.).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.

§ Current address: ChanTest Inc., 4440 Warrensville Center Rd., Cleveland, OH 44118. To whom correspondence may be addressed: Tel.: 216-586-3626; E-mail: rperi@chantest.com.

|| To whom correspondence may be addressed. Rammelkamp Center for Education and Research, R301, MetroHealth Drive, Cleveland, OH, 44109. Tel.: 216-778-5960; E-mail: abrown@research.metrohealth.org.

Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M008445200


    ABBREVIATIONS

The abbreviation used is: Kv, voltage-gated K+.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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