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J. Biol. Chem., Vol. 277, Issue 19, 16376-16382, May 10, 2002

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Characteristics of Brain Kv1 Channels Tailored to Mimic Native Counterparts by Tandem Linkage of  Subunits

IMPLICATIONS FOR K⁺ CHANNELOPATHIES^*

Sobia Akhtar, Oleg Shamotienko, Marianthi Papakosta, Farooq Ali, and J. Oliver Dolly

From the Centre for Neurobiochemistry, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AY, United Kingdom

Received for publication, October 8, 2001, and in revised form, February 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Most neuronal Kv1 channels contain Kv1.1, Kv1.2 , and Kv2.1 subunits, yet the influences of their stoichiometries on properties of the ()₄()₄ variants remain undefined. cDNAs were engineered to contain 0, 1, 2, or 4 copies of Kv1.1 with the requisite number of Kv1.2 and co-expressed in mammalian cells with Kv2.1 to achieve "native-like" hetero-oligomers. The monomeric (Kv1.1 or 1.2), dimeric (Kv1.1-1.2 or 1.2-1.2), and tetrameric (Kv1.1-(1.2)₃) constructs produced proteins of M_r ~62,000, 120,000, and 240,000, which assembled into ()₄()₄ complexes. Each  cRNA yielded a distinct K⁺ current in oocytes, with voltage dependence of activation being shifted negatively as the Kv1.1 content in tetramers was increased. Channels containing 1, 2, or 4 copies of Kv1.1 were blocked by dendrotoxin k (DTX)_k with similarly high potencies, whereas Kv(1.2)₄ proved nonsusceptible. Accordingly, Kv1.2/2.1 expressed in baby hamster kidney cells failed to bind DTX_k; in contrast, oligomers containing only one Kv1.1 subunit in a tetramer exhibited high affinity, with additional copies causing modest increases. Thus, one Kv1.1 subunit largely confers high affinity for DTX_k, whereas channel electrophysiological properties are tailored by the content of Kv1.1 relative to Kv1.2. This notable advance could explain the diversity of symptoms of human episodic ataxia I, which is often accompanied by myokymia, due to mutated Kv1.1 being assembled in different combinations with wild-type and Kv1.2.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Voltage-gated K⁺ channels are involved in the maintenance of resting membrane potential and control of action potential frequency and threshold of excitation (1). Members of the Shaker-related subfamily (Kv1) are sialoglycoprotein complexes (M_r ~400,000) consisting of four transmembrane channel-forming  subunits (Kv1.1-1.6) and four cytoplasmic regulatory  (Kv_1-3) proteins (Ref. 2; reviewed in Ref. 3). Heterologously expressed Kv1 members assemble via their N-terminal domain (NAB) (4) into homo- or heteromultimeric channels with distinct electrophysiological and pharmacological properties (3, 5, 6), although the subunit stoichiometries in the plasmalemma used for the recordings have not been determined. Co-expression of Kv₁ or Kv₃ with Kv1  subunits accelerates inactivation of the K⁺ currents (2, 7), whereas Kv₂ increases surface expression (8-10).

Authentic Kv1 channel proteins were first identified (11) using  and k/dendrotoxin (DTX)¹; DTX is less discriminating and inhibits Kv1.2 > Kv1.1 > Kv1.6 currents, whereas DTX_k is more stringent and specifically blocks Kv1.1 channels (12). Sequential immunoprecipitation, using subunit-specific antibodies, and affinity chromatography on immobilized DTX and/or DTX_k (13-16) unveiled a limited repertoire of subtypes, ranging from Kv(1.2)₄ to those containing Kv1.1/1.2, Kv1.1/1.2/1.6, Kv1.2/1.3/1.4/1.6, or Kv1.1/1.2/1.3/1.4. Also, data from immunocytochemistry and immunological analysis established that Kv1.1, Kv1.2, and Kv2.1 are the most abundant subunits found together in channel complexes (14, 15, 17, 18). These subunits are co-localized in the juxta-paranodal region of the nodes of Ranvier, as well as in the axons and terminals of cerebellar basket cells of rat brain (19). Thus far, heterologous expression of Kv1  and  subunits has failed to mimic the characteristics of neuronal K⁺ currents, highlighting the need to reproduce native K⁺ channels. Additionally, certain neurological conditions are associated with changes in Kv1 channel multimers. For example, an increase in the number of DTX binding sites occurs in demyelinated brain plaques from patients with multiple sclerosis (20), whereas the content of DTX and DTX_k acceptors is decreased in hippocampus from aging patients or those with Alzheimer's disease (21). Furthermore, several mutations in Kv1.1 are associated with human disorders (e.g. episodic ataxia I and myokymia) (1, 22, 23), whereas mutations in certain other genes can distort expression and localization of Kv1.1 and 1.2, thereby inducing abnormal phenotypes (e.g. mouse strains Trembler and Shiverer) (24).

Herein,  subunits were tandem-linked to recreate subtypes with predefined stoichiometries for Kv1.1 and 1.2 and to quantitatively relate subunit composition to channel properties. Functional K⁺ channels containing different numbers of Kv1.1 co-assembled with Kv1.2 in the presence of Kv2.1 were generated using Semliki Forest virus (SFV), yielding oligomers resembling those prevalent in neurons. Electrophysiological recording of their respective K⁺ currents in oocytes and analysis of the binding of ¹²⁵I-labeled DTX_k and DTX to transfected mammalian cells revealed that varying proportions of Kv1.1 and 1.2 could subtly influence the biophysical and pharmacological properties of expressed channels. Such systematic profiling should allow identification of K⁺ channel counterparts in neurons and their altered phenotypes in diseases.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Construction of Monomeric, Dimeric, and Tetrameric cDNAs-- All constructs were incorporated into put2pA for high expression in Xenopus oocytes (9). Monomeric cDNAs were prepared by PCR amplification of rat Kv1.1 and 1.2 subunits, using the respective primer pairs (a) 5'-CCCTCGAGCCACCATGGCGGTGATG-3' and 5'-CTGGTCGACTTTTTAAACATCGGT-3' and (b) 5'- ACTCCTCGAGCACCATGGCAG-3' and 5'-AATAGGTCGACATCAGACATCAGT-3' to introduce XhoI and SalI sites (underlined) with pAKS Kv1.1 or 1.2 cDNA as template (a gift from Prof. O. Pongs). After digestion, the products were ligated into put2pA similarly cleaved. The Kv1.2-1.2 construct was obtained by joining together the two cDNAs via the 5'-untranslated region of the Xenopus -globin gene. For the first position in this tandem, Kv1.2 was amplified from pAKS using primers 5'-CTGCAACTAGTATGACGGTGATGTCAGGGG-3' and 5'-CAACTCGAGATCAGTTAACATTTTGGTAA-3' to remove the stop codon and introduce SpeI and XhoI sites (underlined). After digestion, the PCR product was ligated into put2pA vector cut with XbaI and XhoI to generate put2pA Kv1.2 (without stop codon). To introduce the second constituent of the dimer, a His₆ tag was inserted at the C terminus of Kv1.2, and its initiation codon was removed by PCR to give put2pA Kv1.2 (+His₆, ATG). The latter was amplified using the primer pair 5'-TTTGTCGACACTCAGAAAGAAACGCTC-3' (sense) and 5'-CGTAATACGACTCACTATAGGGC-3' (antisense). After digestion with SalI (introduced by sense primer) and EcoRI (present downstream of coding sequence), the fragment was subcloned into put2pA Kv1.2 (without stop codon) cut with XhoI and EcoRI to generate put2pA Kv1.2-1.2 (+His₆, ATG, 16-amino acid linker, and a stop codon). put2pA Kv1.1-1.2 was designed on the same principle. The coding sequence of rat Kv1.1 was PCR-amplified from pAKS Kv1.1 cDNA using the primer pair 5'-CTGCAACTAGTATGACGGTGATGTCAGGGG-3' (sense) and 5'-CTGGTGCTTCTCGAGAACATCGGTCAGGAG-3' (antisense) to exclude the stop codon and introduce SpeI and XhoI sites (underlined). The digested product was ligated into put2pA (XbaI and XhoI) to generate put2pA Kv1.1 (without stop codon). This was cleaved (XhoI and EcoRI), and the PCR-amplified Kv1.2 (+His₆, ATG) fragment was subcloned downstream of Kv1.1 cDNA, after cutting with SalI and EcoRI. put2pA Kv1.1-(1.2)₃ was obtained by joining Kv1.2-1.2 downstream of Kv1.1-1.2 after manipulations of the two constructs. A His₆ sequence and stop codon were removed from the C terminus of the heterodimer and ligated in-frame to homodimer after deletion of ATG from the first Kv1.2 cDNA to yield put2pA Kv1.1-(1.2)₃.

To prepare a Kv1.2_N-1.1_C chimera, the N-terminal region of Kv1.1 (487 bp) was replaced with the equivalent domain of Kv1.2 (475 bp). put2pA Kv1.1 and 1.2 plasmids were digested with HindIII (outside the coding region) and SacI (487 or 475 bp downstream of Kv1.1 and 1.2, respectively). The isolated larger (~4500 bp from put2pA Kv1.1) and smaller (~475 bp from put2pA Kv1.2) fragments were ligated to yield put2pA Kv1.2_N-1.1_C. Every construct made above was verified by restriction digestion and dideoxy DNA sequencing. For expression in the SFV system, all Kv1 put2pA constructs were subcloned into pSFV1 vector, employing HindIII and BglII sites to cut out the cDNA fragments that were blunt-ended before ligation with pSFV1 vector (10). cRNAs for each Kv1 construct (in put2pA and pSFV1) and pSFVH1 (plasmid encoding viral packaging proteins) were prepared as described previously (10).

Electrophysiological and Biochemical Analysis of Recombinant K⁺ Channels-- Oocytes were isolated from mature Xenopus laevis females (Xenopus I, Blades) and injected with Kv1.1 or 1.2 cRNA, as described by Main et al. (25). K⁺ currents were recorded after 72 h, using a two-microelectrode voltage clamp amplifier (TEC-03; NPI) as described previously (26) during voltage steps to +20 mV from a holding potential of 80 mV. DTX_k was applied by superfusion (at a rate of 5 ml/min), and its concentration increased cumulatively. Addition of tetraethylammonium chloride (0.1-100 mM) was made with substitution of NaCl to maintain ionic strength. A membrane fraction from the injected and noninjected oocytes was analyzed by SDS-PAGE.

Channels were expressed in BHK cells and analyzed in the native state by gel filtration, or by SDS-PAGE and Western blotting as described previously (10). Saturable binding of ¹²⁵I-labeled DTX and DTX_k to intact cells expressing the various constructs was measured in triplicate under established conditions (10) by rapid filtration through GF/F glass microfiber filters that had been presoaked in 0.5% (v/v) polyethyleneimine. The radioactivity associated with the washed filters was quantified by -radiation counting; data presented (±S.E.) were analyzed using the Graph Pad software (Prism 3.0) based on a one-site model.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Voltage Activation of K⁺ Channel Subtypes Prominent in Neurons Is Varied by Predetermining Their Content of Kv1.1 and Kv1.2 Subunits-- Subunit stoichiometries were predefined by linking their cDNAs in an open reading frame (Fig. 1), using a 16-amino acid sequence from 5'-untranslated region of the Xenopus -globin gene. In this way, the expressed Kv1.2-1.2 or Kv1.1-1.2 should assemble into the desired tetrameric proteins, Kv(1.2-1.2)₂ and Kv(1.1-1.2)₂, respectively, whereas the third construct would yield a channel consisting of one Kv1.1 subunit and three Kv1.2 subunits. Biochemical evidence was obtained for formation of the expected oligomers in oocytes, to validate the data subsequently obtained from electrophysiological analysis of the K⁺ currents. Injection into oocytes of cRNAs for the putpA Kv1 constructs followed by Western blotting demonstrated that Kv1.1 and Kv1.2 cRNAs were translated into single subunits of M_r ~60,000-64,000, whereas the dimers and tetramer gave proteins with M_r ~120,000 and M_r 240,000, respectively (Fig. 2A). A two-electrode voltage clamp was used to establish the effects of varying the ratio of Kv1.1 and Kv1.2 in expressed tetramers. Because Kv2.1 has no appreciable effect on activation of Kv1.1 and Kv1.2 channels (26), it was omitted. Oocytes injected with equivalent amounts of cRNAs encoding each of the constructs yielded 1-10 µA K⁺ currents after 72 h, establishing that the expressed proteins were inserted into the plasmalemma as functional channels. Kv1.1, Kv1.2, Kv1.1-1.2, Kv1.2-1.2, and Kv1.1-(1.2)₃ gave outward noninactivating K⁺ currents with characteristic voltage dependences (Fig. 2, B and C). Kv1.2 and Kv1.2-1.2 (homodimer) K⁺ currents exhibited a similar voltage dependence of activation (Table I) (threshold = 45 to 40 mV) and half-maximal activation voltage (V_1/2 = 15.1 and 16.97 mV); the slope (k) values of 10.8 and 8.3, respectively, confirmed the near-identical activation kinetics. Therefore, the linker does not exert a significant influence. Kv1.1 elicited a fast-activating, slow-inactivating K⁺ current that had a more negative activation threshold (60 to 50 mV) than Kv1.2 (Fig. 2, B and C) and V_1/2 = 30.8 mV; these values for the homomeric channels are sufficiently different to allow them to be distinguished reliably. The heterodimer Kv1.1-1.2 cRNA yielded a current that had properties distinct from either parent (Fig. 2, B and C); interestingly, this showed a greater resemblance to Kv1.1 than Kv1.2 current (Table I), with an activation threshold between 55 and 50 mV and a V_1/2 = 26.5 mV. Despite this, the Kv1.1-1.2 K⁺ current proved much less susceptible to blockade by external tetraethylammonium (IC₅₀ = 100 mM) than Kv1.1 (IC₅₀ < 1 mM). A delayed rectifying K⁺ current was observed with Kv1.1-(1.2)₃ (Fig. 2B), activating at a threshold of 55 to 50 mV (Table I); also, its V_1/2 fell between that for the channels made from Kv1.1-1.2 and Kv1.2-1.2 or Kv1.2 constructs (Fig. 2C; Table I), and had a slope similar to the other channels. Thus, Kv1.1 exerts a more dominant influence on K⁺ channel activation, with V_1/2 shifting negatively upon increase in the number of Kv1.1 subunits; on the other hand, their slope factors (k), an indication of activation kinetics, are similar, as expected, because the slope values for the parents are close.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Schematic representation of K+ channels translated from the different constructed cDNAs. A, the putative membrane topology of the expressed chimeric protein translated from a cDNA construct made by fusing the N-terminal region of Kv1.2 (1-475 bp) to the coding sequence of Kv1.1 from which the corresponding region had been deleted (1-486 bp). The dotted line represents the N terminus of Kv1.2, whereas the solid line and barrels, which illustrate the six transmembrane domains, are from Kv1.1. B, cDNAs encoding the homodimer and heterodimer were prepared by linking in tandem the C terminus of Kv1.1 or Kv1.2 (without stop codon) with the N terminus of Kv1.2 via a hydrophilic linker containing 16 amino acids (DTQKETLNFGRSTLEI), whereas the heterotetramer was made by linking the C terminus of heterodimer (without stop codon) to the N terminus of homodimer. The circles show the possible relative positions of the Kv1.1 () and Kv1.2 () subunits in tetramers after translation from each construct. The bars represent linker; noncovalent associations between subunits in tetramers are not depicted.

View larger version (42K): [in this window] [in a new window]   Fig. 2.   Analysis of molecular and electrophysiological properties of the different K+ channels expressed in oocytes A, Xenopus oocytes were injected with the individual cRNAs (~5 ng) and incubated at 16 °C for 48 h, and their crude membranes (lanes 1-3 and 6-8), along with synaptic membranes (lane 4), were subjected to SDS-PAGE (left panel, 9% gel; right panel, 6% gel). After transfer onto polyvinylidene difluoride, the membranes were blocked with 5% (w/v) dried milk before overnight incubation with monoclonal anti-Kv1.2 (lanes 1-5; 1:1000 dilution) or rabbit polyclonal anti-Kv1.1 (lanes 6-8; ~1 µg IgG/ml) and detection with the ECL system. Lane 1, Kv1.2-1.2; lane 2, Kv1.1-1.2; lane 3, Kv1.2; lane 6, Kv1.1-1.2; lane 7, Kv1.1; lane 8, Kv1.1-(1.2)3. Neither Kv1.2 (lane 5) nor Kv1.1 (data not shown) was detectable in the controls injected with water. B, K+ currents were recorded from oocytes 72 h after injection with cRNAs encoding Kv1.1 (I), Kv1.2 (II), Kv1.2-1.2 (III), Kv1.1-1.2 (IV), or Kv1.1-(1.2)3 (V). Voltage dependence of activation was measured by voltage pulses of 400 ms in duration, from a holding potential of 80 mV to +40 mV in 10-mV increments (VI). C, activation curves plotted, using a simple Boltzmann function, as the mean conductances (± S.E.) for each construct (Kv1.1 (), 1.2 (), 1.2-1.2 (), 1.1-1.2 (), and 1.1-(1.2)3 ()) gave the V1/2 and slope values (k) in Table I.

Recreation of Recombinant K⁺ Channels with / Subunit Stoichiometries Mimicking Major Subtypes in Brain-- To generate adequate amounts of the recombinant channels for biochemical analysis, pSFV1 Kv1.1, Kv1.2, Kv1.1-1.2, or Kv1.1-(1.2)₃ was expressed in BHK cells to generate four oligomers representing the majority of possible combinations of the most abundant subunits found in central neurons. The expression level was elevated by inclusion of Kv2.1, which promotes cell surface targeting; to obtain adequate quantities of the poorly expressed Kv(1.1)₄ (10), the N-terminal part was replaced with an analogous moiety of Kv1.2 that regulates the efficiency of assembly (see "Introduction"). This construct gave increased surface expression in BHK cells (~2-fold relative to the unmodified Kv1.1), yielding a subunit of the expected M_r ~60,000-62,000 on immunoblotting (Fig. 3A). The dimer was twice this size and was recognized by both anti-Kv1.1 and anti-Kv1.2 antibodies (Fig. 3, AC), confirming the presence of both  subunits. Kv1.1-(1.2)₃ construct gave a protein of the expected molecular weight (240,000) that was also reactive with anti-Kv1.1 and anti-Kv1.2 antibodies (Fig. 3, B and C). Thus, the dimer and tetramer cRNAs were correctly and fully translated, without any detectable proteolytic breakdown products; importantly, the channels were correctly assembled and inserted into the plasmalemma and functional (Table II; detailed later). Direct evidence for the formation of / subunit oligomers was provided by the oligomeric sizes of the channels extracted from BHK cells in nondenaturing detergent obtained from gel filtration on Superose 6HR (Fig. 3D); the similar elution position for the monomer, dimer, and tetramer expressed with Kv2.1 gave an apparent molecular weight for the oligomer-detergent complex of ~515,000 (Fig. 3D, inset). Because this value is very similar to the size for Kv(1.2)₄(2.1)₄ (10), it can be concluded that all the Kv1 constructs produced proteins that assembled into tetramers containing four  and four  subunits, as observed for neuronal K⁺ channel complexes (27).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Analysis at the subunit and oligomeric levels of the K+ channels expressed using SFV. Cells (~50 µg of protein) infected with SFV encoding chimeric Kv1.1 (A, lane 1), dimeric Kv1.1-1.2 (A, lane 2; B and C, lane 1), or tetrameric Kv1.1-(1.2)3 constructs (B and C, lane 2) were subjected to SDS-PAGE using 9% (A) or 6% gels (B and C) for better resolution and detected as described in Fig. 1A using the antibodies specified. D, another sample was detergent solubilized and chromatographed (10). Fractions were dot-blotted onto polyvinylidene difluoride membrane, with visualization as described in Fig. 1A using anti-Kv1.1 and anti-Kv1.2 IgGs; blots of the peak fractions are shown (inset). The column was calibrated with thyroglobulin, ferritin, catalase, and -amylase; the arrow indicates the common elution position of the K+ channels.

View this table: [in this window] [in a new window]   Table II Binding of 125I-DTXk or 125I-DTX to various K+ channels co-expressed with Kv2.1 in BHK cells and the toxins' mutual antagonism

Homomeric Kv1.1 and Kv1.2 Channels Show Different Affinities for DTX_k and DTX; Their K⁺ Currents Exhibit Corresponding Susceptibilities to DTX_k-- BHK cells expressing Kv(1.2)₄ proved unable to bind ¹²⁵I-DTX_k (Table II); accordingly, the K⁺ current generated in oocytes was insensitive to blockade by 100 nM DTX_k (Table I). In contrast, Kv(1.1)₄ displayed high affinity for ¹²⁵I-DTX_k (Fig. 4A; Table II), consistent with its K⁺ current in oocytes being inhibited by low concentrations of DTX_k (Table I). An avid interaction was reaffirmed for the latter channel by the K_i values of DTX_k competing for the binding of ¹²⁵I-DTX_k (Fig. 4G) and ¹²⁵I-DTX (Fig. 4I). Comparison of the binding of both toxins to Kv(1.1)₄ showed that ¹²⁵I-DTX displayed a 12-fold lower affinity than ¹²⁵I-DTX_k (Table II). On the other hand, ¹²⁵I-DTX exhibited a ~5-fold higher affinity for Kv(1.2)₄ than Kv(1.1)₄ (Table II), in agreement with DTX blocking Kv1.2 K⁺ current with greater potency than Kv1.1 or Kv1.6 (5).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   125I-DTXk and 125I-DTX binding to intact BHK cells expressing the constructed K+ channels: competition with DTXk and DTX. Suspensions of BHK cells expressing chimeric Kv(1.1)4 (A and D), Kv(1.1-1.2)2 (B and E), or Kv1.1-(1.2)3 (C and F) were incubated with 125I-DTXk (AC) or 125I-DTX (DF) at 22 °C for 45 min. In AF, nonsaturable binding () was determined in the presence of 1 µM of the requisite unlabeled toxin and subtracted from the total () to give the saturable component (). For the competition experiments (GI), cells expressing chimeric Kv(1.1)4 (), Kv(1.1-1.2)2 (), and Kv1.1-(1.2)3 () were incubated with 1 nM 125I-DTXk in the absence (B0) and presence of DTXk (G) or DTX (H). In I, 1 nM 125I-DTX was used with Kv(1.1)4 (n), Kv(1.1-1.2)2 (s), and Kv1.1-(1.2)3 () channels, with or without DTXk. Values for nonsaturable binding, measured in the presence of 1 µM DTXk or DTX, have been subtracted from the means (± S.E.) of triplicate values plotted.

One Kv1.1 Subunit in K⁺ Channel Oligomers Confers Near Maximal DTX_k Binding Affinity and Sensitivity of Their K⁺ Currents to Blockade-- Decreasing the number of Kv1.1 subunits in oligomers from four to one caused minimal reduction in their affinity for ¹²⁵I-DTX_k; the K_D values dropped only 2- and 3-fold, respectively, for Kv(1.1-1.2)₂ and Kv1.1-(1.2)₃ (Table II). Likewise, the modest changes in the K_i values (Fig. 4G) for DTX_k antagonizing ¹²⁵I-DTX_k binding to the latter oligomers relative to that for Kv(1.1)₄ support the above deduction. These binding data were corroborated by the DTX_k sensitivities of the channels when expressed in oocytes; the IC₅₀ values of the Kv(1.1-1.2)₂ and Kv1.1-(1.2)₃ K⁺ currents are only 2- and 9-fold lower than that for Kv(1.1)₄ (Table I). Thus, based on both toxin binding and functional blockade, one copy of Kv1.1 is adequate to bestow near maximal affinity for DTX_k and susceptibility to inhibition (see "Discussion").

Moreover, behavior of the channels in binding ¹²⁵I-DTX gave a similar trend with no major difference in the K_D or K_i values upon increasing the number of copies of Kv1.2 in the ()₄()₄ multimers (Table II). Likewise, the similar K_i values for DTX antagonizing ¹²⁵I-DTX_k binding to Kv(1.1-1.2)₂ and Kv1.1-(1.2)₃ (Fig. 4H) revealed that two Kv1.2 subunits are sufficient for binding DTX with high affinity. Because DTX has a lower affinity for Kv(1.1)₄ than DTX_k, it proved significantly less potent in antagonizing ¹²⁵I-DTX_k binding, but this was increased substantially (14-34-fold) when Kv1.2 subunits were introduced to the channels (Fig. 4H). As expected, DTX_k was less effective in displacing ¹²⁵I-DTX from Kv1.2-containing multimers than Kv(1.1)₄ (Fig. 4I).

Finally, it is noteworthy that not only can the two toxins discriminate channel subtypes, but the B_max values for DTX were 2-3-fold higher than for DTX_k in the same batch of BHK cells (Table II; see "Discussion").

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Successful Recreation of the Most Abundant Kv1 Heteromultimers in Mammalian Neurons-- Kv1.1-, Kv1.2-, and Kv2.1-containing oligomers predominate in brain (13, 17); Kv(1.2)₄ is also present, but Kv1.1 always occurs in association with other members (14, 15). Due to their abundance and functional importance (see "Introduction"), we profiled the characteristics of those with several different proportions of Kv1.1 and Kv1.2 to encompass the subtypes found in neurons. All the linked Kv1 subunits were expressed in both amphibian and mammalian cells as single, intact proteins without premature translation or degradation products. Their functionality was documented by the K⁺ currents recorded after expression in oocytes, with characteristics matching those expected. When co-expressed with Kv2.1, each channel co-assembled into ()₄()₄ complexes and was inserted correctly into the plasma membrane of BHK cells, as established from measurement of high affinity binding of DTX_k and DTX, which require  subunits to be assembled into tetramers (28).

Increasing the Number of Kv1.1 Subunits in a Tetramer Gave Commensurate Changes in the Voltage Dependence of Activation of the K⁺ Currents-- The tetramers containing varying ratios of Kv1.1 and Kv1.2 gave slowly inactivating, outward currents with distinct voltage dependences of activation that differed from either parent. The V_1/2 values were slightly skewed toward that of Kv1.1, which may be due to the effect of Kv1.1 upon activation (29, 30). However, the V_1/2 values also clearly reflect the influence of both parental subunits. Establishing the presence of both Kv1.1 and Kv1.2 in this way in the expressed channels constructed by tandem linkage of subunits in the oligomers afforded exploitation of the predetermined stoichiometries for the subsequent toxin block and binding studies.

A Single Kv1.1 Subunit in Tetramers Containing Kv1.2 Creates High Affinity Functional Interaction with DTX_k-- Both saturable binding and competition analysis using intact BHK cells and inhibition of the K⁺ currents in oocytes confirmed that one Kv1.1 subunit in an oligomer is enough to confer a high affinity interaction with DTX_k and blockade of the currents. This conclusion from measurements on defined populations of native-like subtypes accords with a deduction from functional studies on biochemically uncharacterized channels, namely, that a single toxin-sensitive subunit can give K⁺ currents susceptibility to DTX_k homologues (6). Mutagenesis of DTX_k has shown that two domains, 3₁₀ helix and -turn, that are 14 Å apart contribute to interaction with possibly two adjacent channel subunits (31). Notably, the 3₁₀ helix is essential for both high affinity binding of DTX_k to Kv1.1 and precluding its interaction with other subunits; in contrast, the -turn seems less important for recognition of Kv1.1 (31). Hence, it was suggested that the latter region could interact with a different subunit of the K⁺ channels in synaptic membranes; in the present study, this would be another Kv1.1 or Kv1.2. Thus, the slightly lower DTX_k affinity for Kv(1.1-1.2)₂ and Kv1.1-(1.2)₃ compared with Kv(1.1)₄ may be attributed to a reduction in the number of sites for interaction with the 3₁₀ domains and/or a decrease in the affinity of -turn for the adjacent subunit (i.e. Kv1.2 instead of Kv1.1). With two copies of Kv1.1 and Kv1.2, a significant increase in DTX_k affinity was not observed, possibly because of its access being restricted due to the two Kv1.1 in some of the oligomer being positioned diagonally rather than adjacently (Fig. 1B).

Notably, a single high affinity site for DTX_k was observed regardless of the number of Kv1.1 subunits, whereas synaptic membranes show a high affinity and low affinity binding site (31). The retention of high affinity for DTX_k by Kv1.1 channels with up to three insensitive Kv1.2 subunits provides good evidence for the lower affinity site being due to multimerization with other subunits (e.g. Kv1.4, Kv1.3, or Kv1.6) known to be associated with Kv1.1 (13-15, 19) that may result due to steric hindrance. Because Kv(1.1)₄ does not exist in normal human brain (14), and a majority of native K⁺ channels containing Kv1.2 also have Kv1.1 (50%) (13, 17), the latter must represent the bulk of the higher affinity DTX_k sites (31) and thus could explain the overlapping location of DTX_k and DTX acceptors in rat brain (32).

Kv1.1 Channels Apparently Have More Sites for DTX than DTX_k-- The B_max for DTX binding sites was always 2-3-fold higher than that for DTX_k in the same batches of cells. This intriguing finding can possibly be explained by structural elements of DTX_k, DTX (96% homologous to DTX_k), and DTX. Similar residues (e.g. Lys-3/6 and Lys-26) in the two domains (3₁₀ helix and -turn), equivalent to those important for DTX_k recognition of rat Kv1.1-containing channels (31, 33), have also been identified from scanning mutagenesis and thermodynamic mutant cycle analysis in DTX (34). Mutation of the ShaKv1.1 channel revealed that DTX binds at some distance from the pore, involving Lys-3 and Arg-10 among other residues; these, with Lys-26, form a triangle whose vertices are 20 Å apart. This distance would allow interaction with adjacent subunits through Lys-3, Arg-10 (34), and/or Lys-26 (31). However, mutations in the Shaker chimeric channel did not yield evidence for a contribution of Lys-26 to the strong interaction, possibly because it binds residues not substituted that form part of the DTX acceptor site (35, 36). On the other hand, residues at the N terminus of DTX are most influential for Kv1 interaction (37) and are equivalent to the essential amino acids in the 3₁₀ helix of DTX_k, although DTX lacks such a secondary structural feature; this could underlie its less stringent specificity. Furthermore, the contributions of -turn residues in DTX are less pronounced than that for DTX_k (33, 37). Based on the collective findings, DTX_k appears to interact with two Kv1 subunits, whereas DTX requires predominantly one interactive domain; hence, twice as many DTX molecules might be able to bind each tetramer, or perhaps not all the channels are folded perfectly and therefore cannot accommodate the stringent requirements for DTX_k binding.

Functional Properties of the Recombinant Channels in Relation to Those of Neuronal K⁺ Currents in Health and Disease-- Because Kv1.1 and 1.2 subunits exhibit distinct properties, it is not surprising that changing their ratios resulted in K⁺ currents with unique electrophysiological and pharmacological properties. The profiling of their characteristics ought to help molecular entities to be ascribed to the native K⁺ channels, a feat not feasible to date. Thus, properties of the recombinant channels were compared with the two types of DTX-sensitive, sustained K⁺ currents recorded in neuronal cells. Our data for Kv(1.1-1.2)₂ and Kv1.1-(1.2)₃ indicate that they resemble a DTX-sensitive, low-threshold current (I_DS) in various neurons that activates within 50 to 60 mV and prevents repetitive firing (38, 39). Because Kv1.1 and Kv1.2 are the major subunits known to give sustained outward K⁺ currents that are highly sensitive to DTX, their combinations must be responsible for such current phenotypes. Gold et al. (40) have described a similar current (I_Kit) in rat sensory neurons that may relate to a DTX_k-susceptible current found in the same preparation (41); as with the currents we observed, I_Kit activates at low thresholds and is fully activated by +20 mV. From the results herein, it seems that one Kv1.1 subunit in an oligomer with Kv1.2 would be sufficient to give such voltage sensitivity and DTX_k susceptibility; moreover, the slowly inactivating nature of the currents excludes the presence of Kv1.4 and thereby implicates either Kv1.1/1.2 or Kv1.1/1.2/1.6 (15).

These properties derived from channels encompassing most combinations of Kv1.1/1.2 give new insights into the molecular basis of the symptoms seen in patients suffering from episodic ataxia I/myokymia (22, 23). Because we demonstrated herein that these channels' biophysical parameters show gradual changes proportional to their content of Kv1.1 subunits, and a variety of human mutations are known to alter the properties of Kv1.1 homomers (23), the diversity of abnormalities in different cases supports the existence of several hetero-oligomeric combinations of mutated and wild-type Kv1.1, together with Kv1.2; note that Kv(1.1)₄ does not occur in human neurons (14). Such subtypes would exist in different locations, neurons, or compartments (e.g. nerve terminals, axons, and so forth) where each normally serves a pivotal role; thus, a spectrum of abnormalities in individual patients is likely to be due to different stoichiometries of Kv1.2, Kv1.1, and a variant that could be mutated at one of several residues (22, 23). Importantly, the major advance accomplished herein will allow elucidation of the modified properties of all these channel forms constructed by tandem linking Kv1.1, its distinct mutants, and Kv1.2 in the various stoichiometries, followed by co-expression with Kv2.1. Although this strategy has already been found to be informative for dimers of normal and mutated Kv1.1 (42), creating the authentic / combinations should prove much more pertinent.

	ACKNOWLEDGEMENT

We thank Dr. M. Main at Glaxo-Smith-Kline for help with some recordings from oocytes.

	FOOTNOTES

^* This work was supported by a Wellcome Trust grant (to J. O. D.) and a BBSRC CASE studentship in cooperation with Cambridge Drug Discovery (to F. A.).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: Dept. of Biological Sciences, Imperial College of Science, Technology and Medicine, Exhibition Rd., South Kensington, London SW7 2AY, United Kingdom. Tel.: 44-20-75945243; Fax: 44-20-75945312; E-mail: o.dolly@ic.ac.uk.

Published, JBC Papers in Press, February 21, 2002, DOI 10.1074/jbc.M109698200

	ABBREVIATIONS

The abbreviations used are: DTX, dendrotoxin; SFV, Semliki Forest virus; BHK, baby hamster kidney.

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