Mutations in the Kvβ2 Binding Site for NADPH and Their Effects on Kv1.4*

Kvβ2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kvβ2 binds NADP+, and it has been suggested that Kvβ2 is an oxidoreductase enzyme (1). To investigate how this function might relate to channel modulation, we made point mutations in Kvβ2 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 Kvβ2 with Kvα1 channels. To characterize the Kvβ2 mutants functionally, we coinjected wild-type or mutant Kvβ2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kvβ2 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 Kvβ2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kvβ2 subunits with Kv1.4. Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kvβ2 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 Kvβ2 may play a role in the processing of Kv1.4.

cell surface dendrotoxin binding site of Kv1.2 (9). The N terminus of Kv␤1.x (␤1.1, ␤1.2, and ␤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 Kv␣ subunits. The Kv␤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 Kv␤ subunits shares a remarkable structural homology to aldo-keto reductases (10). The crystal structure of the conserved core of Kv␤2 subunit depicts a 4-fold symmetrical TIM barrel structure with the bound cofactor NADP ϩ (1). The structure shows that residues Ser-188 and Arg-189 are important for the orientation of the nicotinamide ring of NADP ϩ and that Asp-85 and Tyr-90 are the putative catalytic site residues of Kv␤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 Kv␤ subunits function as oxidoreductases and, if so, what their physiological substrates may be.
In this study, we investigated the contribution of these residues to Kv␤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 Kv␤2 Mut1 and the orientation mutant (S188A, R189L) is referred to as Kv␤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 Kv␤2 increased Kv1.4 expression by enhancing its trafficking to the cell surface. Mutations at either site eliminated the ability of Kv␤2 to enhance Kv1.4 expression. However, mutant Kv␤2 subunits still interacted with Kv1.4 to increase the rate of inactivation. Thus, the dual effects of Kv␤2 regulation of Kv1.4 were shown to be independent by mutating residues involved in NADP ϩ binding.

EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis-Site-directed mutations (D85A, Y90F mutant 1; S188A, R189L mutant 2) were made in Kv␤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 Kv␤2 cDNAs were subcloned into the pCR3 vector (Invitrogen), which we modified to contain a poly(A) ϩ tail and a unique NruI site for linearizing the constructs prior to cRNA synthesis.
Analysis  1 The abbreviation used is: Kv, voltage-gated K ϩ .
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 Ϫ ) and leucine (leu Ϫ ) after growth for 2-3 days at 30°C. Representative colonies were replated on trp Ϫ /leu Ϫ medium to allow for direct comparison of individual colonies. Transcription of the reporter gene, lacZ, was tested by a ␤-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/l Kv1.4 and 50 ng/l Kv␤2 or Kv␤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 CaCl 2 ⅐2H 2 O, 100 methanesulfonic acid, 2 MgCl 2 ⅐6H 2 O, and 10 HEPES, pH 7.3. All chemicals were purchased from Sigma. We used 50 mM K ϩ 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 Ϯ 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 l/oocyte) in buffer containing 0.25 mM sucrose, 10 mM HEPES 1 mM EGTA, 2 mM MgCl 2 ⅐6H 2 O plus 1 g/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 ϫ g for 10 min to remove debris, and the internal membranes were pelleted by centrifugation at 50,000 ϫ 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-Kv␤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 ϫ g for 10 min to remove debris, and the total membranes were pelleted by centrifugation at 50,000 ϫ g for 60 min.

Interaction of Kv1.4 N Terminus with Kv␤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 Kv␤2 with the Kv1.4 N terminus, as evidenced by activation of the lacZ reporter gene (Fig. 1).
Effect of Kv␤2 Mutants on Kv1.4 Currents-Kv␤2 has been reported to increase both the amplitude and the rate of inactivation of Kv1.4 currents. We investigated the effects of Kv␤2 mutations on Kv1.4 currents by coexpressing cRNAs encoding Kv1.4 with wild-type or mutant Kv␤2 in Xenopus oocytes. Six days after injection, we observed that Kv␤2 increased Kv1.4 current amplitude by 1.6-fold at ϩ80 mV (compare Fig. 2,  panels A and B). The current amplitude measured at ϩ80 mV was 15.35 Ϯ 0.78 A (n ϭ 9) (Fig. 2B) in oocytes expressing a combination of Kv1.4 and Kv␤2 compared with 9.55 Ϯ 0.88 A (n ϭ 7) for oocytes expressing Kv1.4 alone (Fig. 2A). The in-crease in current was manifest at all potentials above threshold (Fig. 3A). When Kv␤2 mutants were examined, the expressionenhancing effect of Kv␤2 on Kv1.4 was abolished. In fact, coexpression with Kv␤2 mutants produced a decrease in the amplitude of Kv1.4 current, and the decrease occurred at all potentials above threshold (Fig. 3A). At ϩ80 mV, mutations in the NADPH oxidoreductase catalytic site (D85A, Y90F) decreased currents by 63.3% to 4.46 Ϯ 0.24 A (n ϭ 5) (Fig. 2C), whereas mutations in the NADPH docking site (S188A, R189L) reduced currents by 36.6% to 6.05 Ϯ 0.45 A (n ϭ 6) (Fig. 2D). The double mutant in which all four residues were simultaneously disrupted also showed a reduction in current ampli-  (n ϭ 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 Kv␤2 or Kv␤2 mutants (p Ͻ 0.01, one-way analysis of variance) were statistically significant.
Yeast two-hybrid data indicated that the mutant Kv␤ subunits were still able to interact with the Kv1.4 N terminus (Fig.   FIG. 3.

Mutations in NADPH docking and oxidoreductase catalytic sites do not significantly alter the biophysical properties of Kv␤2. A, current-voltage relationship of currents recorded from
Xenopus oocytes. Whole cell currents were evoked from a holding potential of Ϫ80 mV by 125-ms depolarizing step pulses to ϩ80 mV in 10 mV increments (current traces shown in Fig.2, A-D). B, coexpression of Kv1.4 with Kv␤2 or Kv␤2 mutants did not alter the voltage dependence of activation. I/I max ratios were calculated by dividing the peak current at each potential with the maximal current at ϩ80 mV and plotted as a function of potential. C, steady state inactivation levels of Kv1.4 in the presence and absence of Kv␤2 and Kv␤2 mutants were similar. Oocytes were held at Ϫ90 mV and pulsed to Ϫ10 mV in 10 mV increments for 1 s followed by a 450-ms test pulse to ϩ70 mV and repolarization to Ϫ90 mV for 6 s (n ϭ 4). Steady state inactivation curves were generated by normalizing the currents at each potential to the maximal currents at ϩ70 mV. The data were fit to the Boltzman equation I/I max ϭ 1/[1 ϩ exp(V 0.5 Ϫ V)/k)], where V 0.5 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 Kv␤2 and Kv␤2 mutants were fitted with a single exponential function. The time constants were plotted as a function of potential (n ϭ 4). Inactivation time constants are significantly smaller when Kv␤2 or wild-type Kv␤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 Ϫ90 mV, the oocyte was depolarized to ϩ70 mV for 1 s and subsequently repolarized to Ϫ90 mV for time intervals ranging from 100 ms to 2.4 s. This was followed by a 400-ms test pulse to ϩ70 mV (n ϭ 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. Kv␤2 slowed the recovery from inactivation compared with Kv1.4 alone, whereas the Kv␤2 mutants produced an even slower recovery from inactivation. Plasma membrane and internal membrane fractions were isolated 6 days post-injection from Xenopus oocytes injected with Kv1.4 (2 ng/l) alone or coinjected with Kv␤2, Kv␤2 mut1, or Kv␤2 mut2 (all at 50 ng/l). 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-Kv␤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 Kv␤2 increased Kv1.4 expression in the plasma membrane fraction, whereas both of the Kv␤2 mutants decreased Kv1.4 protein levels in the internal as well as the plasma membrane fractions. 1). As a further test of association, we examined Kv1.4 currents for evidence of increased inactivation produced by association with Kv␤2 subunits. In an earlier study (8), we had shown that Kv␤2, although absent the inactivation peptide of Kv␤1.x, increased the rate of N-type inactivation of Kv1.4. Normalized current traces of Kv1.4 when coexpressed with either Kv␤2 or Kv␤2 mutants showed that wild-type and mutant Kv␤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 ϩ80 mV, the mean inactivation time constants for Kv1.4, Kv1.4ϩ ␤2, Kv1.4 ϩ ␤2 mut1, and Kv1.4 ϩ ␤2 mut2 were 49.55 Ϯ 2.3, 25.52 Ϯ 0.37, 21.67 Ϯ 0.18, and 22.32 Ϯ 0.28, respectively. Neither wild-type nor mutant Kv␤2 subunits altered the voltage dependence of activation (Fig. 3B) or steady state inactivation (Fig. 3C)  Kv␤2 Enhances Trafficking of Kv1.4 to the Plasma Membrane-Our electrophysiological data showed that the expression-enhancing effect of Kv␤2 on Kv1.4 was distinct from its effects on the biophysical properties of the channel. The enhancement of Kv1.4 expression by Kv␤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 Kv␤2s (Fig. 3B). To determine whether coexpression of Kv␤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 ϳ78-kDa band; and a mature, glycosylated, 105-kDa band, which resolved as a doublet in some experiments (Fig. 4). Coexpression with Kv␤2 did not alter the amounts of Kv1.4 protein in the total membrane fraction. However, coexpression with either of the Kv␤2 mutants did show a reduction in total Kv1.4 protein.
Because Kv␤2 did not increase total Kv1.4 protein levels, we explored the possibility that Kv␤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 Kv␤2 (Fig. 5, right). As expected, only the mature, fully glycosylated ϳ105-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 Kv␤2 (Fig. 5, left).
Consistent with decreased currents, coexpression with Kv␤2 mutants showed a reduction of Kv1.4 in both the plasma and internal membrane fractions (Fig. 5). Both wild-type and mutant Kv␤2 subunits were detected in both the internal and plasma membrane fractions, providing further evidence for the interaction of mutant Kv␤2 subunits with Kv1.4. Although equal amounts of cRNAs were injected for each Kv␤ cDNA, we consistently observed a lower level of expression of Kv␤2 mutant 1. It is not known whether this finding reflects an increased degradation of mutant protein, decreased synthesis, or a combination of both. DISCUSSION Kv␤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 Kv␤2 at the orientation site for NADPH (Mut2) or the putative catalytic site for aldo-keto reductase activity (Mut1) abolished the ability of Kv␤2 to increase Kv1.4 current without changing the ability of Kv␤2 to increase inactivation. The latter result, together with the persistent yeast two-hybrid interactions, showed that the mutant Kv␤2 subunits were still permanently associated with Kv1.4 subunits. However, the Kv␤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 Kv␤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 Kv␤2 (1) and the assembly of Kv␤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 Kv␤2 is important for the processing and trafficking of Kv1.4