Coupling of Voltage-dependent Potassium Channel Inactivation and Oxidoreductase Active Site of Kvbeta Subunits*

Robert BähringDagger , Carol J. Milligan§, Vitya VardanyanDagger , Birgit EngelandDagger , Ben A. Young§, Jens DannenbergDagger , Ralph WaldschützDagger , John P. Edwards§, Dennis Wray§, and Olaf PongsDagger

From the Dagger  Institut für Neurale Signalverarbeitung, Zentrum für Molekulare Neurobiologie der Universität Hamburg, Hamburg, Germany and the § Insitute for Pharmacology, Department of Physiology, University of Leeds, Leeds LS2 9JT, United Kingdom

Received for publication, January 18, 2001, and in revised form, April 6, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

The accessory beta  subunits of voltage-dependent potassium (Kv) channels form tetramers arranged with 4-fold rotational symmetry like the membrane-integral and pore-forming alpha  subunits (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell. 90, 943-952). The crystal structure of the Kvbeta 2 subunit shows that Kvbeta subunits are oxidoreductase enzymes containing an active site composed of conserved catalytic residues, a nicotinamide (NADPH)-cofactor, and a substrate binding site. Also, Kvbeta subunits with an N-terminal inactivating domain like Kvbeta 1.1 (Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej, D. N., Dolly, O., and Pongs, O. (1994) Nature 369, 289-294) and Kvbeta 3.1 (Heinemann, S. H., Rettig, J., Graack, H. R., and Pongs, O. (1996) J. Physiol. (Lond.) 493, 625-633) confer rapid N-type inactivation to otherwise non-inactivating channels. Here we show by a combination of structural modeling and electrophysiological characterization of structure-based mutations that changes in Kvbeta oxidoreductase activity may markedly influence the gating mode of Kv channels. Amino acid substitutions of the putative catalytic residues in the Kvbeta 1.1 oxidoreductase active site attenuate the inactivating activity of Kvbeta 1.1 in Xenopus oocytes. Conversely, mutating the substrate binding domain and/or the cofactor binding domain rescues the failure of Kvbeta 3.1 to confer rapid inactivation to Kv1.5 channels in Xenopus oocytes. We propose that Kvbeta oxidoreductase activity couples Kv channel inactivation to cellular redox regulation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Shaker-related voltage-gated potassium (Kv)1 channels are assembled from membrane-integral pore-forming Kvalpha subunits associated with auxiliary cytoplasmic Kvbeta subunits (4, 5, 6). The membrane topology of Kvalpha subunits shows six membrane-spanning segments, S1-S6, a pore-forming loop structure between S5 and S6, and cytoplasmic N and C termini. A tetramerization domain resides in the N terminus and directs assembly of Kvalpha subunits (7, 8). The tetramerization domain also associates with the auxiliary Kvbeta subunits (9, 10). Crystallographic analysis of Kvbeta 2 tetramers showed that each subunit contains an interface for association with Kvalpha subunits (11) and an oxidoreductase active site (1), but the specific substrate is unknown.

Three Kvbeta genes have been identified: Kvbeta 1, Kvbeta 2, and Kvbeta 3 (12). Kvbeta 1.1 and Kvbeta 3.1 subunits possess an N-terminal inactivating domain that confers rapid N-type inactivation to Kv channels (2, 3). Thus, the association of Kvbeta 1.1 and Kvbeta 3.1 subunits with certain Kv1alpha subunits leads to the expression of rapidly inactivating A-type channels (2, 3). Interestingly, Kvbeta 3.1 confers rapid inactivation to Kv1.5 channels only when coexpressed in mammalian cells (12) but not in Xenopus oocytes (13). By contrast, Kv1.5/Kvbeta 1.1 channels mediate rapidly inactivating currents both in mammalian cells (14, 15) and in Xenopus oocytes (2).

We constructed chimeras between Kvbeta 3.1 and Kvbeta 1.1 to identify possible domains correlated with the apparent lack of function of Kvbeta 3.1 in particular with reference to the N-terminal inactivating domain (2, 3), the interface for association with Kvalpha subunits (11), and the oxidoreductase active site (Ref. 1; see Fig. 1, B and C). The results of our structure-function analysis demonstrate that the failure of Kvbeta 3.1 to confer rapid inactivation to Kv1.5 channels in the Xenopus oocyte expression system is associated with two C-terminal domains of Kvbeta 3.1, which contain structural elements of the oxidoreductase active site. According to the crystal structure of Kvbeta 2, the two Kvbeta 3.1 domains occur in Kvbeta protein regions, which are part of the NADPH cofactor binding pocket and the substrate binding site, respectively. Chimeric replacement of Kvbeta 3.1 residues by Kvbeta 1.1 residues within these domains rescued the Kvbeta 3.1 inactivating activity, and point mutations of Kvbeta 1.1 active site residues attenuated the Kvbeta 1.1 inactivating activity. We propose that Kvbeta oxidoreductase enzymatic activity and the biophysics of Kvbeta inactivating activity are coupled.

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In Vitro Mutagenesis and cRNA Synthesis-- Kvbeta 1.1pAKS (2) and Kvbeta 3.1pAKS (3) were used for Kvbeta 1.1 and Kvbeta 3.1 cRNA synthesis as described previously (16). The cDNAs for the Kvbeta chimeras between rat Kvbeta 1.1 (GenBankTM accession number X70662) and rat Kvbeta 3.1 (GenBankTM accession number X76723) were obtained exploiting appropriate restriction enzyme sites and/or using an overlap polymerase chain reaction (17). The chimeric cDNAs were cloned into the pAKS vector (16). For the construction of the different chimeras the following DNA fragments were joined together (note that numbers in parentheses refer to Kvbeta -cDNA nucleotides): Kvbeta chiA, Kvbeta 3.1-(1-1080) and Kvbeta 1.1-(1000-1546); Kvbeta chiArev Kvbeta 1.1-(1-997) and Kvbeta 3.1-(1075-1599); Kvbeta chiB, Kvbeta 3.1-(390-1260) and Kvbeta 1.1-(1180-1534); Kvbeta chiC, Kvbeta 3.1-(390-1260) and Kvbeta 1.1-(1180-1534); Kvbeta chiD Kvbeta 3.1-(390-1380) and 32 Kvbeta 1.1-(1300-1534); Kvbeta chiE, Kvbeta 3.1-(390-1534) and Kvbeta 1.1-(1453-1534); Kvbeta chiF, Kvbeta 3.1-(390-1450), Kvbeta 1.1-(1370-1453), and Kvbeta 3.1-(1535-1606); Kvbeta chiG, Kvbeta 3.1-(390-1376), Kvbeta 1.1-(1304-1385), and Kvbeta 3.1-(1457-1606); Kvbeta chiH, Kvbeta 3.1-(390-1222), Kvbeta 1.1-(1142-1237), Kvbeta 3.1-(1318-1440), Kvbeta 1.1-(1370-1453), Kvbeta 3.1-(1535-1606); Kvbeta chiI, Kvbeta 3.1-(390-1222), Kvbeta 1.1-(1142-1270), Kvbeta 3.1-(1285-1440), Kvbeta 1.1-(1370-1453), Kvbeta 3.1-(1535-1606).

DNA sequences amplified by the polymerase chain reaction were verified by sequencing using BigDye terminator cycle sequencing kit (PerkinElmer Life Sciences). The sequencing reactions were analyzed on an ABI 377 automated sequencer (PerkinElmer Life Sciences).

Point mutations in rat Kvbeta 1.1 were introduced by site-directed mutagenesis using appropriate mutation primers (17). Polymerase chain reaction products were cloned into Kvbeta 1.1pGEM using a DraIII and a NcoI restriction site. DNA constructs were sequenced prior to use. RNA synthesis was done using the mMessage mMachine in vitro transcription kit according to the manufacturer's protocol (Novagen Inc.).

Recording Techniques and Data Analysis-- Xenopus oocytes were prepared and injected with cRNA, and electrophysiological recordings were made as previously described (18). Briefly, oocytes were injected with 50 nl of a solution containing equal amounts (25 ng) of cRNA for rKv1.5alpha and rKvbeta subunits (wild-type or Kvbeta chi). Deviations from these cRNA concentrations are indicated in the figure legends. Oocytes were then incubated at 20 °C for 24-48 h in multiwell tissue culture plates (one oocyte/well) containing modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.4 mM CaCl2, 7.5 mM Tris-HCl, pH 7.6, 10,000 units/l penicillin, 100 mg/l streptomycin). To record expressed membrane currents, the oocytes were held in a recording chamber (50 µl volume) and continually perfused (2 ml/min) with Ringer's solution (115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 10 mM HEPES adjusted to pH 7.2 with NaOH). Membrane currents were recorded with the two-microelectrode voltage-clamp technique (microelectrodes filled with 3 M KCl) using a Geneclamp 500 amplifier (Axon Instruments), and signals were filtered at 2 kHz. Twenty 10 mV hyperpolarizing steps (1 s duration, 0.5 Hz) were applied and used to remove leak and capacitance currents. To construct current-voltage (I-V) relationships, the cell was held at -80 mV, 1-s duration test depolarizations at 0.1 Hz were applied in 10 mV increments from -70 to +120 mV, and peak and end current amplitudes were measured. I-V curves were fitted with Boltzmann functions of the form I = (V - Vr) × Gmax/(1 + exp((V0.5 - V)/k) where Vr equals -98 mV. The inactivation time course was fit by a sum of two exponentials by the least squares technique. Student's t test was used to test for statistical significance. In some experiments cDNA (vector pcDNA3) for Kv1.5 (25 ng/µl) together with Kvbeta chi cDNA (500 ng/µl) were microinjected into Chinese hamster ovary (CHO) cells. Whole-cell currents were measured with the patch clamp technique on the following day using an EPC9 amplifier and PULSE software (HEKA, Lambrecht, Germany). The extracellular solution contained (in mM) NaCl 135, KCl 5, CaCl2 2, MgCl2 2, HEPES 5, sucrose 10 (pH 7.4, NaOH), and recording pipettes (2-5 MOmega ) were filled with intracellular solution containing (in mM) KCl 125, CaCl2 1, MgCl2 1, EGTA 11, HEPES 10, glutathione 2, K2ATP 2 (pH 7.2, KOH). All experiments were conducted at room temperature (20-22 °C).

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INTRODUCTION
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Kvbeta 1.1 but Not Kvbeta 3.1 Confers Rapid Inactivation to Kv1.5 Channels in Xenopus Oocytes-- Kvbeta 1.1 and Kvbeta 3.1 subunits have functional inactivating domains, but Kvbeta 3.1 confers rapid inactivation to Kv1.5 channels only when coexpressed in mammalian cells (12). In agreement with previous results (13), wild-type Kvbeta 3.1 subunits did not cause rapid inactivation when we coexpressed it with Kv1.5 channels in Xenopus oocytes (Fig. 1A; Table I). Upon depolarization of the oocyte membrane, slowly inactivating outward currents were recorded, and 84% of the initial peak amplitude remained at the end of a 1-s test pulse to +80 mV (Fig. 1, A and D; Table I). By contrast, Kv1.5/Kvbeta 1.1 channels mediate rapidly inactivating currents both in mammalian cells (14, 15) and in Xenopus oocytes (2). In our experiments they decayed to ~13% of the initial maximum current amplitude at the end of a 1 s test pulse to +80 mV (Fig. 1, A and D; Table I). The inactivation time course (Fig. 1A) was fitted with two time constants, tau 1 (13.0 ± 0.9 ms) and tau 2 (75.0 ± 0.5 ms; n = 14; Table I). The fast time constant tau 1 accounted for 90 ± 10% of the total current decay. We examined the structural motifs in Kvbeta 3.1 responsible for its inactivation failure in Xenopus oocytes by constructing chimeras between Kvbeta 3.1 and Kvbeta 1.1. Possible structural determinants correlated with the apparent lack of function of Kvbeta 3.1 may be located in the N-terminal inactivating domain (2, 3), the interface for association with Kvalpha subunits (11) and/or the oxidoreductase active site (Ref. 1; Fig. 1, B and C).


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Fig. 1.   Inactivating activity of Kvbeta 1.1, Kvbeta 3.1, and the chimeric Kvbeta subunits Kvbeta chiA - I when coexpressed with Kv1.5. A, currents were elicited in Xenopus oocytes expressing Kvalpha /beta combinations as indicated on top of each trace. The current traces were recorded using depolarizing steps from a holding potential of -80 mV to a test potential of +80 mV of 1 s. Leak currents have been subtracted. Bars show current and time calibrations. B, bar diagrams show in gray relative positions of domains and, respectively, amino acid residues of Kvbeta 1.1 corresponding to the N-type inactivation domain, the Kvbeta oxidoreductase active site, and the interface for assembly with Kvalpha subunits. C, bar diagrams of chimeric constructs Kvbeta chiA - I (open bars, Kvbeta 1.1; filled bars, Kvbeta 3.1). Numbers on top refer to first and last Kvbeta 1.1 amino acid residue; numbers at bottom refer to first and last Kvbeta 3.1 amino acid residue in each construct. D, mean percentage (I1s / Ipeak) of peak current (Ipeak) remaining after 1 s of inactivation (I1s) for Kv1.5 coexpressed with Kvbeta 1.1, Kvbeta 3.1, and each of the constructs as indicated (experimental protocol as in A; n = 9-20).

                              
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Table I
Summary of gating parameters for Kv1.5 currents in the presence of wild-type and chimeric Kvbeta subunits
The decay phase of outward currents during a 1-s pulse to +80 mV was fit by a double-exponential function, which yielded tau 1, tau 2, and the percentage of the total decay accounted for by tau 1. The amount of inactivation was accessed by the fractional current remaining after 1 s (I1s / Ipeak). V0.5 values and respective slope factors (k) for steady-state activation were obtained as described under "Experimental Procedures." Numbers of oocytes (n) are given for both kinetic analysis and voltage dependence of activation. In all cases 25 ng of Kv1.5 and Kvbeta cRNA was injected per oocyte.

Failure of Kvbeta 3.1 in N-type Inactivation Linked to C-terminal Domains-- We connected in the first chimera the Kvbeta 3.1 N terminus (residues 1-229), which contains the N-terminal inactivating domain and the conserved catalytic residues (1) of the Kvbeta 3 oxidoreductase (Fig. 1B), with the Kvbeta 1 C terminus corresponding to Kvbeta 1.1 residues 223-401 (Kvbeta chiA; Fig. 1C). The N-terminal Kvbeta 3.1 inactivating domain became fully functional when connected to the Kvbeta 1 C terminus (Fig. 1, A and D), in agreement with the previous observation that the Kvbeta 3.1 inactivating domain becomes functional when connected to Kvbeta 2.1 (3). The results obtained with Kvbeta chiA indicated that C-terminal Kvbeta 3.1 domain(s) must be responsible for the lack of functional interaction with Kv1.5 channels. To test this hypothesis, we constructed a reverse chimera (Kvbeta chiArev), in which the Kvbeta 1 N-terminal half was linked to the Kvbeta 3 C-terminal half (Kvbeta 1 residues 1-222 and Kvbeta 3 residues 230-404; Fig. 1B). The Kv1.5/Kvbeta chiArev-mediated currents exhibited inactivation kinetics similar to the ones observed for Kv1.5 with wild-type Kvbeta 3.1, with 80 ± 1% of the maximal current amplitude remaining at the end of a 1-s depolarizing pulse (Fig. 1A; Table I). These results confirmed the idea that the ability of Kvbeta chiA to inactivate Kv1.5 channels was due to the presence of the Kvbeta 1.1 C terminus.

To identify the C-terminal domains responsible for the failure of Kvbeta 3.1 in N-type inactivation we constructed further Kvbeta chimeras. For this purpose we expanded the Kvbeta 3 portion in Kvbeta chiA in a stepwise fashion. Additional replacement of Kvbeta 1.1 residues 223-282 by those of Kvbeta 3.1 (Kvbeta chiB) included the Kvbeta 1 interface domain for assembly with Kvalpha subunits (Ref. 11; Fig. 1, B and C). The respective Kv1.5/Kvbeta chiB channels mediated outward currents with a relatively small reduction in the extent of inactivation (Fig. 1, A and D; Table I). The small reduction would be compatible with a subtle difference in the affinities of Kvbeta 1.1 and Kvbeta 3.1 subunits for the Kv1.5alpha subunits (11). Nevertheless, the results demonstrated that the Kvbeta 3.1 domains for N-type inactivation and the subunit interface for complex formation with Kv1.5alpha subunits were not responsible for the observed inactivation failure of Kvbeta 3.1. The exchange of Kvbeta 1.1 residues by those of Kvbeta 3.1 was extended to residues 283-346 (Kvbeta chiC, -D), which may cover the entire substrate binding site of the Kvbeta oxidoreductase (Ref. 1; Fig. 1, B and C). The respective Kv1.5/Kvbeta chiC and Kv1.5/Kvbeta chiD currents showed a significant reduction in the extent of inactivation (Fig. 1, A and D) because of an increase in tau 2 as well as an alteration in the relative weights of the fast (tau 1) and slow (tau 2) components (Table I). We extended the exchange of Kvbeta 1.1 by Kvbeta 3.1 residues further to residues 347-374 (Kvbeta chiE), which encompassed a part of the Kvbeta oxidoreductase active site that is essential for binding the cofactor NADPH (Ref. 1; Fig. 1, B and C). The resulting Kv1.5/Kvbeta chiE currents showed an ineffective and slow inactivation (Fig. 1, A and D) due to an increase in both tau 1 and tau 2 (Table I). The results suggested that C-terminal Kvbeta 3.1 domains encompassing substrate and cofactor binding sites were responsible for the failure of Kvbeta 3.1 to inactivate Kv1.5 channels.

Inactivating Activity of Kvbeta 3.1 Rescued by Swapping with Kvbeta 1.1 Domains-- With chimeras Kvbeta chiF - I, we tried to rescue the inactivation failure of Kvbeta 3.1 by exchanging Kvbeta 3.1 domains with appropriate Kvbeta 1.1 domains (Fig. 1, B and C). Exchanging Kvbeta 3.1 residues 354-381 for those of Kvbeta 1.1 produced Kvbeta chiF, which conferred to the slowly inactivating Kv1.5 currents a rapid inactivation (Fig. 1, A and D; Table I). By contrast, replacement of Kvbeta 3.1 residues 328-353 by those of Kvbeta 1.1 (Kvbeta chiG) had no significant effect on inactivation (Fig. 1, A and D; Table I). Supplementation of Kvbeta chiF with an additional Kvbeta 1.1 sequence replacing Kvbeta 3.1 residues 278-309 (Kvbeta chiH) did not markedly alter the activity of Kvbeta chiF toward Kv1.5 channels. However, extending the replacement in Kvbeta chiH by an additional 21 amino acids (Kvbeta chiI) generated Kv1.5/Kvbeta chiI currents with rapid and nearly complete inactivation (Fig. 1, A and D; Table I). At the end of the 1-s test pulse to +80 mV the current was reduced to ~6% of the initial maximum current amplitude similar to Kv1.5/Kvbeta 1.1 currents (Fig. 1D). The data demonstrated that the exchange of two C-terminal Kvbeta 3.1 domains (domains I and II in Fig. 1C) by those of Kvbeta 1.1 sufficed to rescue the inactivation failure of Kvbeta 3.1 in Xenopus oocytes. Kvbeta domains I and II provide amino acid residues to the Kvbeta oxidoreductase active site. Domain I contributes to the Kvbeta NADPH cofactor binding site (Ser-325, Gln-329, Glu-332, and Asn-333 in Kvbeta 2.1; Ref. 1). Residues in domain II (Kvbeta 1.1 amino acids 303-323; Fig. 1B and C; see also Fig. 4A) have been proposed to participate in substrate binding in Kvbeta 2 (1).

Voltage Dependence of Kv1.5 Current Activation Affected by Kvbeta Chimeras-- The voltage-gating properties of Kv1.5 channels were affected by the presence of different Kvbeta 1.1/Kvbeta 3.1 chimeras (Table I). This is typically observed upon assembly of Kvalpha with Kvbeta subunits (3, 14, 19-21). Voltages of half-maximal current activation (V0.5 = +17 to +22 mV) and slope factors (k = 19-27 mV) of the conductance-voltage relationships were similar for Kv1.5/Kvbeta 1.1, Kv1.5/Kvbeta 3.1, and Kv1.5/Kvbeta chi currents except for Kv1.5/Kvbeta chiB (V0.5 = +36 mV), Kv1.5/Kvbeta chiC (V0.5 = +38 mV), and Kv1.5/Kvbeta chiF currents (V0.5 = +27 mV) (Table I). Obviously, the distinct inactivation time courses of the various Kv1.5/Kvbeta chi currents at +80 mV, especially the rapid decay of Kv1.5/Kvbeta chiI currents, are not due to an altered voltage-dependent steady-state activation.

Kv1.5/Kvbeta chi Coexpression Mediates A-type Currents in CHO Cells-- We considered the possibility that the structural differences at specific sites between Kvbeta 1.1 and Kvbeta 3.1 lead to improper protein folding and thereby to the observed functional inactivity of the Kvbeta chimeras. Because the wild-type Kvbeta 3.1 is functionally active in CHO cells (12), we tested the activity of certain Kvbeta chimeras in the same expression system. We chose Kvbeta chiE, Kvbeta chiF, and Kvbeta chiG for these experiments because this series exhibited both gain of functional activity (from Kvbeta chiE to Kvbeta chiF) and loss of functional activity (from Kvbeta chiF to Kvbeta chiG) in Xenopus oocytes. When we coexpressed Kv1.5 channels with these chimeras in CHO cells, all the combinations gave rise to rapidly inactivating currents (Fig. 2A). We used a high excess of Kvbeta chi cDNA (20-fold; see "Experimental Procedures") to ensure a high expression level of beta  subunits expected to yield a maximal fraction of Kvalpha /beta complexes (22). Therefore, we also performed control experiments in Xenopus oocytes with a similar excess of Kvbeta 3.1 cRNA over Kv1.5 channel cRNA. The results are shown in Fig. 2, B and C. They were not significantly different from what we obtained in the experiments with equal amounts of alpha  and beta  subunit cRNA (see Fig. 1). The results suggested that improper folding or insufficient protein expression represented the most unlikely explanations for the functional inactivity of Kvbeta chimeras in Xenopus oocytes.


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Fig. 2.   Functionality of Kvbeta 3 subunits depends on the expression system. A, Kv1.5/Kvbeta chi currents recorded with the patch clamp technique in CHO cells. Kvbeta chi cDNA was microinjected at a 20-fold higher concentration than Kv1.5 cDNA. Note that all tested Kvbeta chimeras conferred rapid inactivation to Kv1.5 channels. B, oocyte currents mediated by Kv1.5 and Kv1.5/Kvbeta 3.1 channels. Oocytes were injected with 1 ng of Kv1.5 and 20 ng of Kvbeta 3.1cRNA (1:20 ratio). Despite the high expression level, Kvbeta 3.1 subunits failed to rapidly inactivate Kv1.5 channels in the Xenopus oocyte expression system. Note, however, that the relative current amplitude (in %) remaining at the end of a 1-s pulse was smaller in the presence of Kvbeta 3.1 as shown in C.

Mutations of Putative Catalytic Residues Attenuate Kvbeta 1.1 Inactivating Activity-- Because the differing inactivating activities of Kvbeta 1.1 and Kvbeta 3.1 were correlated to sequence differences in their oxidoreductase active sites, we mutated the putative catalytic residues in Kvbeta 1.1 (Asp-119, Tyr-124, and Lys-152), which are highly conserved in the superfamily of oxidoreductase enzymes (1). We investigated the effect of the mutations on Kvbeta 1.1-mediated N-type inactivation. Coexpression of the mutants Kvbeta 1.1D119A, Kvbeta 1.1Y124F, and Kvbeta 1.1K152A with Kv1.5 at cRNA ratios of 1:1 (Fig. 3A) and 1:5 (Fig. 3C) showed that the respective outward currents did not decay as rapidly as Kv1.5/Kvbeta 1.1 currents (Table II). Each mutation affected rapid inactivation behavior to a different degree. The most marked effect was observed with Kvbeta 1.1D119A, which did not confer rapid inactivation to Kv1.5 channels. However, 15-s test pulses revealed that Kvbeta 1.1D119A did accelerate the slow inactivation of Kv1.5 currents (Fig. 3, B and D), which most likely represents a C-type inactivation (23, 24). As N-type and C-type inactivation are coupled (25), the observed acceleration of Kv1.5 inactivation was probably due to some residual inactivating activity of the Kvbeta 1.1D119A subunit.


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Fig. 3.   Examples of normalized outward currents mediated by Kv1.5 alone or coexpressed with wild-type Kvbeta 1.1 (Kvbeta 1.1wt) or the Kvbeta 1.1 mutants Kvbeta 1.1D119A, Kvbeta 1.1Y124F, and Kvbeta 1.1K152A, respectively, as indicated by the numbers 1-5. Currents were elicited in Xenopus oocytes using depolarizing steps from a holding potential of -80 mV to a test potential of +80 mV in A and B and to a test potential of +60 mV in C and D. Leak currents have been subtracted. Bars indicate time calibrations. Recordings in A and B are from oocytes injected with 3 ng of Kv1.5 and 3 ng of Kvbeta cRNA (1:1 ratio), whereas in C and D 3 ng of Kv1.5 and 15 ng of Kvbeta cRNA were injected (1:5 ratio).

                              
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Table II
Summary of gating parameters for Kv1.5 currents in the absence or presence of wild-type (wt) and point-mutated Kvbeta 1.1 subunits
The decay phase of outward currents during a 1-s pulse to +80 mV was fit by a double-exponential function (tau 1, tau 2, and percentage of the total decay accounted for by tau 1). The amount of inactivation was accessed by the fractional current remaining after 1 s (I1s/Ipeak). The fitting parameters for Kv1.5 alone and for Kv1.5 + Kvbeta 1.1D119A were obtained from 15-s pulses. V0.5 values and respective slope factors (k) for steady-state activation were obtained as described under "Experimental Procedures." The values in brackets are from oocytes, which were injected with a 1:5 ratio of Kv1.5 versus Kvbeta 1.1 cRNA and pulsed to +60 mV for kinetic analysis. In all cases 3 ng of Kv1.5 cRNA was injected per oocyte. Numbers of oocytes (n) are given for both kinetic analysis and voltage dependence of activation.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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It has been shown that rapid N-type inactivation of Shaker Kv channels requires the presence of an N-terminal inactivating domain and of a receptor close to the inner entrance of the Shaker channel pore (4). Upon depolarization the inactivating domain rapidly binds to the receptor and thereby occludes the pore. Kvbeta 3.1, like Kvbeta 1.1, contains a functional inactivating domain (3) and thus may confer rapid N-type inactivation to Shaker type channels. However, the inactivating activity of Kvbeta 3.1 depends on the in vitro expression system; in CHO cells Kvbeta 3.1 confers rapid inactivation to Kv1.5 channels (12) but fails to do so in Xenopus oocytes (3, 13). Our results demonstrate that the lack of function of Kvbeta 3.1 in Xenopus oocytes was correlated with two C-terminal Kvbeta 3.1 domains encompassing the NADPH and substrate binding sites, respectively, of the Kvbeta oxidoreductase active site. Domain I in Fig. 1C and Fig. 4 contributes to the Kvbeta NADPH cofactor binding site (Ser-325, Gln-329, Glu-332, and Asn-333 in Kvbeta 2.1; Ref. 1). Seven of the eight domain I residues that differ between Kvbeta 1.1 and Kvbeta 3.1 (Fig. 4A) are near or at the Kvbeta adenosine-binding pocket (Fig. 4, B and C). Residues in domain II (Kvbeta 1.1 amino acids 303-323; Fig. 4A) have been proposed to participate in substrate binding, in particular Kvbeta 1.1 residue Trp-306 that corresponds to Trp-272 in Kvbeta 2 (1). When domains I and II in Kvbeta 3.1 were replaced by those in Kvbeta 1.1, the resulting Kvbeta 3.1/Kvbeta 1.1 chimeras were able to confer rapid inactivation to Kv1.5 channels in the Xenopus oocyte expression system. The results indicated that the functional activity of Kvbeta 3.1 in Xenopus oocyte could be rescued by replacing Kvbeta 3.1 amino acid residues in the oxidoreductase site by the ones of Kvbeta 1.1. In agreement with the assumption that C-terminal Kvbeta 3 domain(s) are responsible for the observed lack of function, Kvbeta 1.1 was rendered non-functional when the C-terminal half of the protein was replaced by Kvbeta 3.1 sequences.


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Fig. 4.   Localization of domains I and II in the Kvbeta 2 crystal structure. A, alignment of domain I and II sequences of Kvbeta 1.1, Kvbeta 2.1, and Kvbeta 3.1. Numbers at right and left refer to first and last amino acid residue shown. Asterisks indicate amino acid residues that are in close contact with the adenosine moiety of the NADPH cofactor bound in the Kvbeta 2 active site (1). A Black dot marks the Kvbeta 2 amino acid residue proposed to contribute to the substrate binding site (1). Identical amino acid residues are in red. Amino acid residues are given in the single letter code. B, ribbon representation of the three-dimensional structure of Kvbeta 2 tetramer according to Ref. 1. Domains I and II as defined in Fig. 1C are colored red. NADPH cofactor (green) is shown as Corey-Pauling-Koltun model. Domain I residues 325, 329, 332, and 333 and domain II residue 272 are labeled. Side-chains are in stick representation and have been colored magenta. C, Kvbeta monomer to illustrate domain I side-chains in NADPH binding pocket and domain II side-chain in substrate binding site. Model was prepared with the program INSIGHT (Molecular Simulations Inc., San Diego, CA) and based on the Kvbeta 2 structure as available in the Protein Database accession number 1 QRQ.

Three main alternatives may be considered to understand the results: i) Kvbeta 3.1 is not active because the oxidoreductase site is not properly folded; ii) Kvbeta 3.1 activity is inhibited in Xenopus oocytes by an as yet unknown factor; and iii) Kvbeta 3.1 is not active because the Xenopus oocytes do not provide a substrate for the Kvbeta 3.1 oxidoreductase. The results showed that Kvbeta 3.1 and Kvbeta chimeras are functionally active in CHO cells. This demonstrates that active and properly folded Kvbeta 3.1 protein can be expressed in in vitro expression systems. Most likely, translation and folding of Kvbeta 3.1 protein in Xenopus oocytes is not different from that in CHO cells. Therefore, it is unlikely that Kvbeta 3.1 (and the Kvbeta chimeras) is not properly folded when expressed in Xenopus oocytes. The existence of a putative inhibitory factor in Xenopus oocytes, which is specific for Kvbeta 3.1, cannot be rigorously excluded but seems to be also unlikely. Thus, we assume that Kvbeta 3.1 fails to confer rapid inactivation to Kv1.5 channels because its oxidoreductase activity is not functioning in Xenopus oocytes. In agreement with this assumption we find that a replacement of the NADPH and the putative substrate binding domains by those of Kvbeta 1.1 reconstitutes the inactivating activity of Kvbeta 3.1 in Xenopus oocytes.

Because Kvbeta 3.1 oxidoreductase activity is apparently important for conferring rapid inactivation to Kv1.5 channels, we explored the possibility that mutations of catalytic residues in Kvbeta 1.1 (Asp-119, Tyr-124, Lys-152) may attenuate the Kvbeta 1.1 inactivating activity. The catalytic residues are highly conserved among the active sites of oxidoreductases (1), and comparable mutations in established oxidoreductase enzymes have been shown to impair catalytic activity (26, 27). The results showed that the mutations severely affected the ability of Kvbeta 1.1 to confer rapid inactivation to Kv1.5 channels. Although the putative Kvbeta enzymatic activity could not be tested directly, it is likely that the mutations of catalytic residues in Kvbeta 1.1 affected its putative oxidoreductase activity. We propose that Kvbeta oxidoreductase catalytic activity is required for the inactivating activity of Kvbeta 1.1. Thus, manipulations of the putative Kvbeta 1.1 and Kvbeta 3.1 oxidoreductase active sites were correlated with a loss and, respectively, gain of inactivating activity in the Xenopus oocyte expression system.

Although we have not carried out biochemical experiments to test directly the binding of Kvbeta 3.1 to Kv1.5, the effects of Kvbeta 3.1 and the different Kvbeta 1.1/Kvbeta 3.1 chimeras on the voltage-gating properties of Kv1.5 are a clear indication that Kvbeta 3.1 binds to Kv1.5. Changes in the voltage-gating properties of Kv1 channels are typically observed upon assembly of Kvalpha with Kvbeta subunits (3, 14, 19-21). In conclusion, Kvbeta 3.1 assembles with Kv1.5 channels, but the activity of the N-terminal Kvbeta 3.1 inactivating domain is impaired. Apparently, the effects of Kvbeta 3.1 on the voltage-dependent activation of Kv1.5 channels are distinct from those leading to rapid inactivation. This is in agreement with the previous observation that removal of 10 amino acids from the Kvbeta 1.3 N terminus eliminated the inactivation activity but not the voltage shift of activation of Kv1.5 channels (28).

Kvbeta 2 subunits do not have an N-terminal inactivating domain. When coexpressed with Kvalpha subunits, Kvbeta 2 may also alter the voltage-gating properties of Kv channels and, in addition, enhance trafficking of Kv channels to the plasma membrane. In agreement with our results, it has been shown in a recent report (29) that mutating active site residues in Kvbeta 2 did not interfere with its binding to Kv1.4 channels. These Kvbeta 2 mutants still affected the voltage-gating properties of Kv1.4 channels like wild-type Kvbeta 2, but the enhancing effects on Kv1.4 channel surface expression were attenuated. Apparently, the putative Kvbeta oxidoreductase activity is important for distinct aspects of Kvbeta function.

Previously, we have shown that N-type inactivation can be prevented by a NIP-domain in Kv1.6 subunits (15). Now, we show that N-type inactivation of Kv channels may be coupled to the putative Kvbeta 1.1 and Kvbeta 3.1 oxidoreductase activity. This observation indicates that the gating mode of Kv channels linked to N-type inactivation may be regulated by a variety of cellular mechanisms. We propose that the presence of an oxidoreductase activity in Kv channels may couple cellular redox regulation to the gating mode of Kv channels allowing the channels to switch between a rapidly inactivating and a non-inactivating mode. Identifying the Kvbeta oxidoreductase substrate(s) will bring us closer to understanding the cellular function of such potential energetic coupling.

    ACKNOWLEDGEMENTS

We thank S. Maudsley for performing initial experiments, M. Berger and S. Sewing for help with the construction of Kvbeta chiA - E chimeras, D. Clausen for technical help with the figures, and C. Legros for modeling Kvbeta 2.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft (O. P.) and the Wellcome Trust (D. 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.

To whom correspondence should be addressed: Zentrum für Molekulare Neurobiologie der Universität Hamburg, Martinistr. 52, 20246 Hamburg Germany. Tel.: 049-40-2803-5081/5082; Fax: 049-40-42803-5102; E-mail: Pointuri@uke.uni-hamburg.de.

Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M100483200

    ABBREVIATIONS

The abbreviations used are: Kv, voltage-dependent potassium; CHO, Chinese hamster ovary.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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