Modulation of Voltage-Gated K Channels by the Sodium Channel Β1

Modulation of voltage-gated K+ channels by the sodium channel β1 subunit

Hai M. Nguyena, Haruko Miyazakib, Naoto Hoshic, Brian J. Smithd, Nobuyuki Nukinab, Alan L. Goldine, and K. George Chandya,1

Departments of aPhysiology and Biophysics, cPharmacology, and eMicrobiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697; bLaboratory for Structural Neuropathology, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan; and dLa Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia

Edited* by Michael D. Cahalan, University of California, Irvine, CA, and approved September 26, 2012 (received for review June 1, 2012) β – Voltage-gated sodium (NaV) and potassium (KV) channels are critical Here, we examined whether the NaV 1 KV4.x interaction was components of neuronal action potential generation and propaga- specific for this family of KV channels, or whether NaVβ1 could β SCN1b tion. Here, we report that NaV 1 encoded by , an integral interact and modulate the functions of other families of KV chan- subunit of NaV channels, coassembles with and modulates the bio- nels in mammals. We selected three families of KV channels (KV1, physical properties of KV1andKV7 channels, but not KV3 channels, in KV3, and KV7) for these studies. We report that NaVβ1modulates fi β an isoform-speci c manner. Distinct domains of NaV 1 are involved the function of KV1.1, KV1.2, KV1.3, KV1.6, and KV7.2 channels, in modulation of the different KV channels. Studies with channel but not KV3.1 channels, in an isoform-specific manner. Through chimeras demonstrate that NaVβ1-mediated changes in activation the use of chimeric and mutational strategies we identified regions kinetics and voltage dependence of activation require interaction in NaVβ1 and KV channels required for channel modulation, and of NaVβ1 with the channel’s voltage-sensing domain, whereas docking simulations suggest a molecular model of the interaction of changes in inactivation and deactivation require interaction with the two proteins. the channel’s pore domain. A molecular model based on docking

studies shows NaVβ1 lying in the crevice between the voltage-sens- Results ﬁ ing and pore domains of KV channels, making signi cant contacts NaVβ1 Interacts with and Modulates KV1.x Channels. We examined

with the S1 and S5 segments. Cross-modulation of NaV and KV chan- the ability of NaVβ1 to modulate activation kinetics, voltage NEUROSCIENCE β fl nels by NaV 1 may promote diversity and exibility in the overall dependence of activation and deactivation of KV1.x channels control of cellular excitability and signaling. expressed in mammalian cells or in Xenopus oocytes. In Xenopus oocytes, NaVβ1 accelerated KV1.2 activation, shifted the voltage colocalization | gating | voltage-gated ion channels | patch-clamp | dependence of activation in the hyperpolarized direction and sped accessory subunit up the fast component of deactivation (τfast at −60 mV) (Fig. 1 A– C). In mammalian cells, KV1.2 exhibited use-dependent activation equential opening and closing of central nervous system (CNS) (i.e., successive depolarizing pulses progressively increase the am- voltage-gated sodium (Na ) and potassium (K ) channels plitude of the KV1.2 current and speed up activation), a unique S V V β fi mediate the depolarization phase (1) and repolarization phase (2, property of this channel (16). NaV 1signi cantly accelerated KV1.2 τ fi 3), respectively, of neuronal action potentials. Although intrinsic activation ( fast at +40 mV) at the rst pulse and to a lesser extent at the ninth pulse (Fig. 1D). Coprecipitation experiments demon- domains within the NaV α subunits underlie voltage-dependent strated that NaVβ1 (dual-tagged at the C terminus with V5 and gating properties and sodium-specific permeation, five NaVβ sub- His ×)andmouseK 1.2 (FLAG-tagged at the C terminus) coas- units (β1, β1B, β2, β3, β4) assemble with and modulate inactivation 6 V sembled when heterologously expressed in mammalian cells (Fig. kinetics and the voltage dependence of activation and inactivation E α β 1 ). Furthermore, immunostaining experiments on normal mouse of NaV subunits (1, 4). In addition, these NaV subunits function β fi brain showed KV1.2 colocalization with NaV 1 in the axon initial in cell adhesion and contribute to neuronal migration, path nding, segment of neurons in the cerebral cortex (Fig. 1F)butnotinnodes – and fasciculation (4 8). Given their ubiquitous roles and distri- of Ranvier (Fig. S1A). bution in the CNS, Na β subunits play a major role in fine-tuning V KV1.1 and KV1.3 had a different staining pattern and did not of action potential generation, propagation, and frequency. Subtle colocalize with NaVβ1 in normal mouse brain (Fig. S1 B and C). changes to their function have resulted in a range of detrimental However, these channels may colocalize with NaVβ1 in de- neurological diseases in humans such as genetic epilepsy with fe- myelinating diseases where their distribution is altered (17–19). brile seizures plus (GEFS+), Dravet syndrome (severe myoclonic We therefore tested the effect of NaVβ1onKV1.1, KV1.3, and epilepsy of infancy), temporal lobe epilepsy, febrile seizures, and KV1.6 channels (Table S1). NaVβ1 shifted the voltage depen- decreased responsiveness to the anticonvulsant drugs carbamaze- dence of activation of KV1.1 by ∼4 mV in the hyperpolarized pine and phenytoin (7–12). direction and it slowed deactivation (τfast) of the channel without A–C β fi KV channels are important contributors to action potential re- affecting its activation kinetics (Fig. 2 ). NaV 1 signi cantly Xenopus D polarization. KV channels in mammals are encoded by 40 genes accelerated activation of KV1.3 in oocytes (Fig. 2 ) and grouped into 12 subfamilies (KV1–KV12). NaVβ1, which was previously thought to be specific for NaV channels, was recently shown Author contributions: H.M.N. and K.G.C. designed research; H.M.N., H.M., N.H., B.J.S., and to coassemble with and modulate the properties of the KV4.x – β N.N. performed research; B.J.S. and A.L.G. contributed new reagents/analytic tools; H.M.N., subfamily of channels (13 15). In the rodent heart, NaV 1 copre- H.M., N.H., B.J.S., N.N., and K.G.C. analyzed data; and H.M.N., B.J.S., and K.G.C. wrote cipitates with KV4.3, and in heterologous expression systems it the paper. increases the amplitude of the KV4.3 current and speeds up acti- The authors declare no conflict of interest. vation (14, 15). In the rodent brain, NaVβ1 coprecipitates with *This Direct Submission article had a prearranged editor. KV4.2, and in heterologous expression systems it enhances surface Freely available online through the PNAS open access option. expression of the channel and increases current amplitude (13). 1To whom correspondence should be addressed. E-mail: [email protected]. β fi Genetic knockout of NaV 1 in mice prolongs action potential ring This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in pyramidal neurons through its action on KV4.2 (13). 1073/pnas.1209142109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1209142109 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Activation kinetics at +40 mV (A, gray hatched area in pulse protocol represents ﬁrst 30 ms analyzed), voltage dependence of activation (B) (mean ±

SEM), and deactivation (C, gray hatched area in pulse protocol represents last 10 ms analyzed) of KV1.2 channels expressed in Xenopus oocytes in the presence or absence of NaVβ1(n ≥ 7). Only currents of comparable amplitude between channel-alone, channel plus NaVβ1, were selected for analysis. (D)KV1.2 stably expressed in L929 ﬁbroblasts exhibits use-dependent activation when repetitive depolarizing pulses at +40mV are administered at 1-s intervals. Average

normalized current traces show NaVβ1 speeds up activation of KV1.2 at the ﬁrst pulse and to a lesser extent at the ninth pulse (KV1.2, n =6;KV1.2 + NaVβ1, n = 9 cells). (E) Western blot of coprecipitation experiment in transfected HEK cells showing that KV1.2 and NaVβ1 coassemble. (F) Mouse cerebral cortex immunostained for KV1.2 (green; Left) and NaVβ1 (red; Center), showing colocalization in the axon initial segment in the merged image (Right).

in mammalian cells (Fig. S2F) but had no effect on KV1.3’s axon initial segment and at nodes of Ranvier (25). In mammalian voltage dependence of activation or deactivation kinetics (Fig. 2 cells, NaVβ1 coassembled with FLAG-tagged KV7.2 (Fig. 2M), and E and F). NaVβ1 significantly reduced cumulative inactivation of it slowed the channel’s activation at moderate depolarization vol- KV1.3 (Fig. S2 A–C), a unique property of KV1.3 where successive tages (Fig. 2N, Fig. S4) and altered current measured at different 1- depolarizing pulses cause a progressive diminution in the ampli- s prepulse potentials (Fig. 2O). These data together with those tude of the KV1.3 current as channels accumulate in the C-type presented in Fig. 1 and in the literature (13–15) demonstrate that inactivated state (18). His-tagged NaVβ1 complexes with and NaVβ1 modulates KV1.1, KV1.2, KV1.3, KV1.6, KV4.2, KV4.3, and accelerates activation of KV1.3 in mammalian cells and Xenopus KV7.2, but not KV3.1, each in a unique fashion. oocytes (Fig. S2 D–F). NaVβ1 slowed KV1.6’s activation, shifted its β voltage dependence of activation in the depolarized direction, and Distinct Domains of NaV 1 Are Involved in KV Channel Modulation. β significantly increased the amplitude of the KV1.6 tail current, but NaV 1 and myelin zero (P0), the major protein in peripheral nerve had no effect on the channel’s deactivation kinetics (Fig. 2 G–I). myelin (26), are both type-1 membrane proteins that contain an Endogenous K channels in pheochromocytoma (PC12) cells, extracellular Ig fold, a single transmembrane segment, and an in- V A β which include KV1.x channels (20, 21), were also modulated by tracellular C-terminal region (27) (Fig. 3 ). NaV 1 sped up ac- Na β1(Fig. S3). Thus, Na β1’s modulatory effects on K 1 chan- celeration of KV1.2 and KV1.3, whereas P0 slowed down KV1.3 V V V B E nels vary in an isoform-specific manner. activation and had no effect on KV1.2 activation (Fig. 3 and ). Because NaVβ1andP0 have different effects on KV1.2 and KV1.3, β β fi NaV 1 Modulates KV7 Channels but Not KV3 Channels. The KV3 family we used two chimeras of NaV 1andP0 to de ne the regions re- contains four members, KV3.1–KV3.4. KV3.1 is a fast-activating/ quired for modulation of these channels (Fig. 3A). These chimeras fast-deactivating channel that regulates spike frequency in several were previously used to identify NaVβ1 domains required for NaV brain regions (22–24). When expressed heterologously in mam- channel modulation (28). The NaVβ1-P0 chimera contains the malian cells, NaVβ1 had no effect on activation kinetics, voltage extracellular Ig domain of NaVβ1 and the transmembrane and dependence of activation or deactivation of KV3.1 (Fig. 2 J–L). intracellular segments of P0, whereas the P0-NaVβ1 chimera con- fi – The KV7 family contains ve members, KV7.1 KV7.5. KV7.2 tains the extracellular domain of P0 and the transmembrane and and KV7.3 are the main molecular components of the neuronal intracellular segments of NaVβ1 (Fig. 3A). M-current, a noninactivating, slowly deactivating, subthreshold The external Ig domain of NaVβ1 is solely responsible for ac- current that regulates neuronal excitability (25). We examined celeration of KV1.3 activation because the NaVβ1-P0 chimera whether NaVβ1 modulated KV7.2 channels, which are found in the (containing NaVβ1’s external domain) sped up KV1.3 activation

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Fig. 2. Activation kinetics (A, D, G, and J), voltage dependence of activation (B, E, H, and K) (mean ± SEM), and deactivation kinetics (C, F, I, and L)ofKV1.1, KV1.3, KV1.6, and KV3.1 expressed alone (black) or with NaVβ1 (red) in Xenopus oocytes (KV1.1, KV1.3, and KV1.6) or mammalian cells (KV3.1) (n ≥ 5). (M) Western blot of coimmunoprecipitation experiment in transfected HEK cells showing that KV7.2 and NaVβ1 coprecipitate. (N)NaVβ1 slows activation of the KV7.2 current in CHO cells at moderate depolarizing potentials (n = 5). (O) Relative instantaneous current of KV7.2 shows a signiﬁcant difference in the 1-s prepulse potential in the presence of NaVβ1(n =9).

like NaVβ1, but the P0-NaVβ1 chimera (containing NaVβ1’s trans- like NaVβ1 (Fig. 3E), indicating that an intact external Ig domain membrane/intracellular domain) had no effect (Fig. 3B). In sup- is also not sufficient for this modulation. β C port, two GEFS+ mutations (C121W and R85C) that disrupt the Full-length NaV 1slowedKV1.1 deactivation (Fig. 2 ). This modulation requires the transmembrane and/or cytoplasmic re- external Ig domain abolished NaVβ1-mediated acceleration of gion of NaVβ1 because the C121W NaVβ1 mutant was as effective KV1.3 activation (Fig. 3C). The external domain of NaVβ1 was also as full-length NaVβ1 in slowing KV1 deactivation (Fig. S5C). Col- responsible for reducing cumulative inactivation of KV1.3 because lectively, our results demonstrate that distinct NaVβ1 domains are the NaVβ1-P0 but not the P0-NaVβ1 chimera significantly reduced required for modulating different KV channels, each in a channel cumulative inactivation, and the two GEFS+ mutations abolished isoform-specific manner. this modulation (Fig. S5 A and B). Full-length Na β1 accelerated K 1.2 activation but neither V V NaVβ1 Interacts with the Voltage-Sensing and Pore Domains of KV1 chimera recapitulated this modulation, indicating that the entire Channels. We constructed chimeras of KV1.1 and KV1.3 to identify E–G β protein is required (Fig. 3 ). Two NaV 1 GEFS+ mutants channel regions required for NaVβ1-mediated modulation. We with a disrupted external Ig domain accelerated KV1.2 activation chose this pair of channels because of the distinctive modulatory

Nguyen et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 Fig. 3. Schematic representation of NaVβ1 (red), myelin P0 protein (blue), and their chimeras (A). Inset, Extracellular domain of NaVβ1 showing the R85C and C121W epilepsy-causing mutations. (B and E) Effect on KV1.2 and KV1.3 activation kinetics by NaVβ1 versus P0 (B and E), chimeras (C and F), and NaVβ1 mutants (D and G) studied in Xenopus oocytes. Current traces are averages of normalized, representative currents of comparable amplitudes and shown are the ﬁrst 30-ms activating phase of the 200-ms traces.

effects of NaVβ1. NaVβ1 sped up activation and decreased cumu- segment of NaVβ1 lies in the groove between the VSD and PD of lative inactivation of KV1.3, whereas it had no effect on KV1.1’s KV1.2, making significant contacts with the channel’s S1 and S5 M N activation and inactivation (Fig. 2, Table S1). NaVβ1 shifted the segments (Fig. 4 and ). An alignment of the S5 segments β voltage dependence of activation of KV1.1 in a hyperpolarizing shows several key differences between NaV 1-resistant KV3.1 and β A direction and it slowed deactivation (τfast at −60 mV) of KV1.1, but NaV 1-modulated channels (Fig. S8 ). The transmembrane seg- β did not alter these properties in KV1.3 (Fig. 2, Table S1). The ment of NaV 1 makes contact with many of the key S5 residues. β KV1.1–KV1.3 chimera contains the voltage-sensing domain (VSD) Several highly conserved residues on NaV 1 make contact with the β of KV1.1 (S1–S4) and the pore domain (PD) of KV1.3 (S5–P–S6), channel. Leu13 and Leu17 of NaV 1 (transmembrane numbering; B whereas the KV1.3–KV1.1 chimera contains the VSD of KV1.3 and Fig. S8 ) form a pocket into which Phe25 of the KV1.2-S5 segment β the PD of KV1.1 (Fig. S6). Both chimeras produced robust currents binds. Trp20 of NaV 1 packs against Gly18 of the S5 segment, and (Fig. 4 A and B). Cumulative inactivation, a unique property of the Glu24 of NaVβ1 forms hydrogen bonds with the uncapped N-ter- A KV1.3 PD, was exhibited by the KV1.1–KV1.3 chimera but not by minal main-chain amides of the slide helix (Fig. S8 ). Residues of β the KV1.3–KV1.1 chimera (Fig. 4 G and H), indicating that both the S1 segment predicted to interact with NaV 1 include Ala3, chimeras function as predicted. Val7, Leu11, Val15, and Leu19 (S1 numbering; Fig. S8C). Val15’s The VSD of KV1.3 is required for NaVβ1-mediated acceleration of side chain packs against the side chain of Tyr11 of the trans- β KV1.3 activation because the KV1.3–KV1.1 chimera (containing membrane segment of NaV 1. In the S1 segment, KV3 channels fi KV1.3’s VSD) exhibited faster activation in the presence of NaVβ1, differ signi cantly from KV1 channels at position 15 (V/T), from whereas the activation of the KV1.1–KV1.3 chimera (containing Kv4 channels at positions 3 (Y/A), 7 (G/L), 11 (A/L), and 15 (I/T), KV1.3’s PD) was unaffected by NaVβ1(Fig.4C, Table S1). The PD of and from Kv7 channels at positions 3 (Y/A) and 15 (L/T). The Ig β – – KV1.3 is required for NaVβ1-mediated reduction of KV1.3 cumula- domain of NaV 1 forms extensive interactions with the S1 S2, S5 – M N tive inactivation because NaVβ1 reduced cumulative inactivation of P, and P S6 extracellular loops of the channel (Fig. 4 and ). the KV1.1–KV1.3 chimera (containing KV1.3’s PD), but not the Discussion KV1.3–KV1.1 chimera (containing KV1.3’sVSD)(Fig.4G and H). The VSD of KV1.1 is required for the NaVβ1-mediated change The precision of neuronal action potential generation and propa- in the voltage dependence of KV1.1 activation because the gation is controlled by the sequential activation and inactivation of α – KV1.1–KV1.3 chimera (which contains KV1.1’s VSD), but not the NaV and KV channels. In the CNS, four NaV subunits (NaV1.1 β – β KV1.3–KV1.1 chimera (which contains KV1.1’s PD), exhibited NaV1.3, NaV1.6) in tight complex with NaV 1 NaV 4 subunits un- a hyperpolarizing shift in the voltage dependence of activation in derlie the depolarization phase of the action potential. Several the presence of NaVβ1 (Fig. 4 D and E, Table S1). The PD of subfamilies of KV channels are important contributors to action – β KV1.1 is required for NaVβ1-mediated slowing of KV1.1 de- potential repolarization (1 4). NaV 1, although considered to be activation because the KV1.3–KV1.1 chimera (KV1.1’s PD), but a subunit of NaV channels, was recently shown to interact with and – not the KV1.1–KV1.3 chimera (KV1.1’s VSD), showed slower modulate the function of KV4 channels in the heart and brain (13 β deactivation in the presence of NaVβ1 (Fig. 4 J and K). 15). Here, we demonstrate that NaV 1 is a promiscuous protein that To examine whether the interaction between NaVβ1 and the canalsointeractwithandmodulatethepropertiesofKV1(KV1.1, KV1.3’s PD sterically hinders toxin access to the external channel KV1.2, KV1.3, KV1.6) and KV7(KV7.2) channels. Of the channels vestibule, we measured the affinity of the channel for ShK-186, tested, only KV3.1 was not affected by NaVβ1. Modulation occurs in fi – aKV1.3-selective peptide inhibitor (29). ShK-186 blocked KV1.3 in a channel isoform-speci c manner. Through structure function a dose-dependent manner and its potency did not change in the approaches, we identify regions of NaVβ1 and KV channels required β presence of NaVβ1(Fig. S7). This result suggests that the NaVβ1– for NaV 1-dependent modulation, and we provide a model of the β KV1.3 interaction does not alter toxin access to the channel vesti- NaV 1andKV channel complex. β bule. Overall, these results demonstrate that NaVβ1 accelerates NaV 1 coassembles with KV1.2, speeds up its activation, shifts its KV1.3 activation by interacting with the KV1.3’s VSD, affects cu- voltage dependence of activation by 4 mV in the hyperpolarized mulative inactivation of KV1.3 through an interaction with KV1.3’s direction, and slows its deactivation in mammalian cells and in Xenopus β PD, induces a hyperpolarizing shift in the voltage dependence of oocytes. NaV 1andKV1.2 colocalize in the axon initial activation of KV1.1 via an interaction with the KV1.1’s VSD, and segment in the mouse cerebral cortex where the interaction be- β slows deactivation of KV1.1 via an interaction with KV1.1’s PD. tween the two proteins may affect neuronal excitability. NaV 1 shifts the voltage dependence of activation of KV1.1 in the hyper- Molecular Modeling. Docking of full-length NaVβ1 with the KV1.2 polarized direction, slows deactivation (τfast), and has no effect on homotetramer resulted in a model in which the transmembrane activation kinetics. NaVβ1 accelerates activation of KV1.3, abolishes

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Fig. 4. Activation kinetics (A and B), voltage dependence of activation (D and E; mean ± SEM), cumulative inactivation (G and H), and deactivation of KV1.1– KV1.3 and the KV1.3–KV1.1 chimeras (J and K), expressed alone (black) or with NaVβ1 (red) in Xenopus oocytes. Schematic showing interaction between NaVβ1 and domains within the channel chimeras (C, F, I, and L). Current traces shown are averages of normalized currents of selective sample currents of comparable

amplitudes. Side view (M) and view from the extracellular side of the membrane (N) of the model of the complex of KV1.2 with NaVβ1. The channel monomers are colored in four different shades of blue, NaVβ1 in tan, and residues that are lipid exposed on the S1 and S5 segments and that differ in character to equivalent residues in other KV channel families are displayed as green and orange spheres, respectively.

cumulative inactivation (accelerates recovery from C-type in- We used chimeras of NaVβ1andP0 to define regions in NaVβ1 activation) and has no effect on its voltage dependence of activation responsible for KV channel modulation. The NaVβ1 domains re- or deactivation kinetics. NaVβ1 slows KV1.6 activation, shifts its quired for channel modulation varied in an isoform-specific manner. voltage dependence of activation in a depolarized direction, and The external Ig domain of NaVβ1 is solely responsible for modu- significantly increases the amplitude of the KV1.6 tail current lation of KV1.3, whereas the entire NaVβ1 protein is required to without affecting its deactivation kinetics. Another KV channel modulate KV1.2 and KV1.1. Chimeras of KV1.3 and KV1.1 were found in the axon initial segment in the brain, KV7.2, coassembles used to identify channel regions required for NaVβ1-mediated with NaVβ1 in mammalian cells and its activation is slowed at modulation. The PD (S5–P–S6) of KV1.3 is required for NaVβ1’s moderate depolarizing potentials. However, NaVβ1 does not alter modulation of cumulative inactivation, whereas its VSD (S1–S4) activation or deactivation kinetics, or the voltage dependence of is essential for NaVβ1-mediated acceleration of KV1.3 activation. activation of the KV3.1 channel. The PD of KV1.1 is required for NaVβ1’s slowing of deactivation,

Nguyen et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 whereas the VSD is essential for the NaVβ1-mediated hyper- results provide a mechanism for coordinated control of two im- polarizing shift in KV1.1’s voltage dependence of activation. portant classes of ion channels that underlie neuronal excitability. The model of NaVβ1dockedwithKV1.2 permits an interpretation of the isoform-specific properties of the different channels at the Materials and Methods Electrophysiology. Whole-cell patch-clamp and two-electrode voltage-clamp level of sequence-specific interactions. All of the NaVβ1-sensitive techniques were used for analysis of mammalian cells and Xenopus oocytes, KV channels contain Leu at position 17 in place of a Phe in KV3 channels (numbering based on Fig. S8A), an aromatic residue (Phe respectively. Details are provided in SI Materials and Methods. or Tyr) at position 22 in place of Ile, and an Ile at position 29 in place Expression Plasmids, Immunohistochemistry, Coprecipitation, Western Blots, and of Leu. The increased bulk and hydrophobic interactions of KV3’s ’ Molecular Modeling. Expression plasmids, immunohistochemistry, coprecipi- Phe17, and the decreased bulk of KV3 sIle22,andthelossofhy- tation, Western blots, and molecular modeling are described in SI Materials drophobic interactions between the side chain of Phe/Trp22 (in and Methods. KV1, KV4, KV7 channels) with NaVβ1, likely impacts the interaction between KV3andNaVβ1. Trp20 of NaVβ1 packs against Gly18 of Cell Culture and Transfections. L929 cells stably expressing KV1.2, KV1.3, and K 3.1, CHO cells, and PC12 cells were maintained in standard DMEM con- the S5 segment; this residue is Leu in the KV3 family. The increased V bulk of the side chain of Leu likely impacts the interaction between taining 10% (vol/vol) heat-inactivated FCS (Summit Biotechnology), 4 mM L- glutamine, 1 mM sodium pyruvate, and 500 μg/mL G418 (Calbiochem) as the proteins. Phe25 of the S5 segment of KV1.2 fits into a pocket β described in SI Materials and Methods. Transient transfections were carried formed by two Leu residues (Leu13, -17) of NaV 1; the smaller Ala out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s present in KV3 channels may pack less well into this pocket and instructions. After 24–30 h, transfection efficiency was assessed by fluores- thereby affect binding affinity. The side chain of Val15 of the S1 cence microscopy (Olympus). segment of KV1.2 packs against the side chain of Tyr11 of the transmembrane domain of NaVβ1. Substitution of the hydrophobic ACKNOWLEDGMENTS. We thank Dr. M. K. Mathew (National Centre for Biological Sciences) for expression constructs of K 1.1, K 1.2, and K 1.6 Val15 with Thr in KV3 will impact the interaction. Therefore, V V V ’ β channels; Radit Aur (University of California, Irvine) for preparing the KV3.1 s resistance to NaV 1 modulation may be because residues in oocytes; Dr. Lori Isom (University of Michigan) for myelin P ,His-V5-tagged β 0 6 its S1 and S5 hinder optimal interaction with NaV 1. NaVβ1, NaVβ1-P0 chimera, P0-NaVβ1 chimera; and Dr. Jeffrey Calhoun (Uni- In conclusion, our results provide a structural basis for a NaVβ1- versity of Michigan) for verifying the sequence of the chimeras. A generous allocation of computational resources from the Victorian Life Science Com- mediated regulation of KV1, KV4, and KV7 channels. By modulating β ’ puting Initiative is acknowledged (to B.J.S.). This work was supported CNS potassium currents, NaV 1 s importance in channel gating- by National Institutes of Health Grants NS48252 (to K.G.C.), NS048336 (to modulation is extended beyond its influence on NaV currents. Our A.L.G.), and NS067288 (to N.H.).

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