Crystallographic insights into sodium-channel PNAS PLUS modulation by the β4 subunit John Gilchrista,1, Samir Dasb,c,1, Filip Van Petegemb,c,2, and Frank Bosmansa,d,2 aDepartment of Physiology and dSolomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and bDepartment of Biochemistry and Molecular Biology and the cLife Sciences Institute, University of British Columbia, Vancouver, V6T 1Z3 Canada Edited by Baldomero M. Olivera, University of Utah, Salt Lake City, UT, and approved November 4, 2013 (received for review July 31, 2013) Voltage-gated sodium (Nav) channels are embedded in a multicom- Moreover, the notion that the β4 subunit shapes the overall ponent membrane signaling complex that plays a crucial role in pharmacological sensitivities of the Nav channel signaling com- cellular excitability. Although the mechanism remains unclear, plex remains unexplored. However, altered ligand interactions β-subunits modify Nav channel function and cause debilitating dis- maybeexploitedtodetectthepresenceofβ4 in normal or orders when mutated. While investigating whether β-subunits also pathological conditions (29). Here, we investigated whether fl β in uence ligand interactions, we found that 4dramaticallyalters β-subunits influence Nav channel sensitivity to molecules iso- toxin binding to Nav1.2. To explore these observations further, we lated from animal venom and discovered that β4 can drastically β solved the crystal structure of the extracellular 4 domain and iden- alter the response of the neuronal Nav1.2 isoform to spider and fi 58 ti ed Cys as an exposed residue that, when mutated, eliminates scorpion toxins that target paddle motifs within Nav channel voltage the influence of β4 on toxin pharmacology. Moreover, our results sensors. To elucidate the machinery underlying this observation, we suggest the presence of a docking site that is maintained by a cyste- solved the crystal structure of the extracellular β4 domain and found β 58 ine bridge buried within the hydrophobic core of 4. Disrupting this a Cys-containing binding interface that is involved in Nav channel bridge by introducing a β1 mutation implicated in epilepsy reposi- modulation of toxin pharmacology by β4. Remarkably, dismantling 58 tions the Cys-containing loop and disrupts β4 modulation of Nav1.2. the strictly conserved internal cysteine bridge in β4 by introducing Overall, the principles emerging from this work (i) help explain tissue- a β1 mutation implicated in epilepsy (30) does not preclude protein dependent variations in Nav channel pharmacology; (ii) enable the folding and trafficking to the membrane. However, conformational mechanistic interpretation of β-subunit–related disorders; and (iii) changes induced by the mutation perturb the 58Cys-containing loop PHYSIOLOGY provide insights in designing molecules capable of correcting aber- and disrupt β4 interaction with Nav1.2, in turn altering the func- β rant -subunit behavior. tional and pharmacological properties of the larger Nav channel signaling complex. voltage-gated sodium channel | beta4 subunit | ProTx-II | X-ray structure | disease mutations Results β-Subunits Shape Nav Channel Pharmacology. Although the influence of β-subunits on ion channel gating is well documented (5), little is oltage-gated sodium (Nav) channels play a key role in cel- Vlular communication by manipulating the transmembrane known about their ability to manipulate the pharmacological voltage gradient to encode and propagate vital information rapidly sensitivities of Nav channel signaling complexes (29, 31, 32). To investigate the extent to which these versatile glycoproteins modify over long distances (1). Consequently, mutations that modify Nav channel activity underlie debilitating neurological diseases, mus- ligand interactions, we applied seven toxins from spider, scorpion, sea anemone, and wasp venom (ProTx-I, ProTx-II, TsVII, AaHII, cular disorders, and pain syndromes (2, 3). Typically, Nav channels are part of a membrane-embedded signaling complex that involves various integral membrane proteins (4). The significance of this Significance environment for proper channel function is highlighted by di- vergent Nav channel responses to changes in membrane voltage Voltage-gated sodium (Nav) channels are members of a large when expressed in native tissues or in heterologous systems. complex that plays a crucial role in rapid electrical signaling β-Subunits are prominent members of the Nav channel signaling throughout the human body. As prominent members of this β complex but do not contribute to the ion-conducting pore (5). complex, -subunits modify Nav channel function and cause Instead, they are multifunctional single-transmembrane segment debilitating disorders when mutated. Collectively, the func- glycoproteins that (i) modulate the gating properties of voltage- tional and crystallographic results reported in this work uncover gated ion channels; (ii)regulateNav channel trafficking and ex- intricate interactions of these elements within the Nav-channel pression levels; and (iii) promote cell adhesion and migration (5– signaling complex and establish a key role for β-subunits in 14). Of the four known β-subunits and their splice variants (15–20), shaping Nav1.2 pharmacology. An important concept emerging β4 is unique in that it enables resurgent current, a feature that from our results is that β-subunits provide exciting opportuni- ties for designing new therapeutic strategies to correct their renders certain Nav channel isoforms capable of high-frequency firing in excitable tissues (21). Moreover, aberrant behavior of the abnormal behaviors. ubiquitously expressed β4 subunit has been implicated in long-QT Author contributions: J.G., S.D., F.V.P., and F.B. designed research, performed research, syndrome (LQTS) (22), LQTS-associated Sudden Infant Death analyzed data, and wrote the paper. Syndrome (23), atrial fibrillation (24), Huntington’s disease (25), The authors declare no conflict of interest. and prostate cancer (26), possibly through dysregulation of the Nav channel signaling complex. β4 also is targeted by β-andγ-secretase This article is a PNAS Direct Submission. enzymes from the amyloidogenic pathway, a recent observation Data deposition: Crystallography, atomic coordinates, and structure factors reported in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes that suggests a potential contribution of this particular subunit to 4MZ2 and 4MZ3). ’ the development of Alzheimer s disease (27). 1J.G. and S.D. contributed equally to this work. Despite accumulating evidence supporting an important con- 2To whom correspondence may be addressed. E-mail: fi[email protected] or tribution to neuronal excitability and various health disorders [email protected]. (28), fundamental questions about the molecular mechanisms This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. underlying β4 interaction with Nav channels remain unanswered. 1073/pnas.1314557110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1314557110 PNAS Early Edition | 1of9 Downloaded by guest on October 1, 2021 LqqIV, ATX-II, and β-PMTX) as well as two drugs (lidocaine and Nav1.2 and to inhibit channel opening (34, 35). However, in the β ambroxol) to Xenopus oocytes expressing the neuronal Nav1.2 presence of 4, Nav1.2 is dramatically less inhibited, suggesting isoform and determined potential changes in ligand susceptibility that β4 may prevent ProTx-II from interacting with one or more induced by the presence of each of the four β-subunits. We un- of its receptor sites (Fig. 1B). Second, the structurally unrelated covered multiple conditions in which Nav1.2’s sensitivity to a par- scorpion toxin TsVII promotes Nav channel opening by prefer- ticular toxin was modified by β-subunits, whereas sensitivity to entially interacting with the voltage sensor in domain II and sta- neither drug was significantly affected (Fig. 1, Fig. S1,andTable bilizing it in an activated state (34, 36). Without β-subunits present, S1). For example, the sea anemone toxin ATX-II interacts exclu- 500 nM TsVII causes Nav1.2 to open at more negative voltages, sively with the Nav1.2 domain IV voltage sensor to inhibit fast although the maximal conductance of the channel is not affected. inactivation, resulting in a large increase in inward sodium ion flow In contrast, TsVII greatly decreases Nav1.2 maximal conductance (33). When β2 is present, 100 nM ATX-II still prevents Na 1.2 from with a smaller shift in activation voltage when β4 is present (Fig. v C β fl inactivating rapidly; however, the peak sodium current increases 1 ), raising the possibility that this -subunit exerts an in uence on only marginally (Fig. S1). A similar effect is seen when 100 nM of the domain II voltage sensor. Next, we crystallized the extracellular β fl the domain IV-targeting scorpion toxin LqqIV is applied to Na 1.2 4 domain to explore the mechanisms underlying the in uence of v β coexpressed with β1 [binding site identification is given in Fig. S2 this particular -subunit on Nav1.2 pharmacology. (34)]. In this instance, however, LqqIV also shifts the steady-state The β4 Subunit Structure Reveals an Exposed Cysteine. Despite their inactivation curve to more positive potentials (V from −57 mV 1/2 impact on the functional and pharmacological properties of Na to −48 mV; P ≤ 0.001), thereby increasing channel availability to v channels, tertiary structural information about β-subunits has