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Voltage-Gated Sodium Channel (Nav ) Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins David Shayaa, Mohamed Kreirb, Rebecca A. Robbinsc, Stephanie Wonga, Justus Hammona, Andrea Brüggemannb, and Daniel L. Minor, Jr.a,c,d,e,f,1 aCardiovascular Research Institute, cDepartments of Biochemistry and Biophysics and dCellular and Molecular Pharmacology, eCalifornia Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158-9001; fPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and bNanion Technologies GmbH, Gabrielenstrasse 9, D-80636 Munich, Germany Edited by Richard W. Aldrich, University of Texas at Austin, Austin, TX, and approved June 14, 2011 (received for review April 30, 2011) Many voltage-gated ion channel (VGIC) superfamily members microscopy studies (19 Å) that have suggested some general contain six-transmembrane segments in which the first four form features of NaVs purified from eel electric organs (11) but that a voltage-sensing domain (VSD) and the last two form the pore lack the resolution for detailed mechanistic insight. Study of bac- domain (PD). Studies of potassium channels from the VGIC super- terial NaVs provides a simplified system for understanding basic family together with identification of voltage-sensor only proteins aspects of both NaV and CaV function and holds promise as a have suggested that the VSD and the PD can fold independently. template for guiding small molecule modulator development (6). Whether such transmembrane modularity is common to other Although there have been ongoing efforts to produce bacterial VGIC superfamily members has remained untested. Here we show, NaV samples that can be used for biochemical and structural using protein dissection, that the Silicibacter pomeroyi voltage- studies (12–14), these have yet to achieve the multi-milli-gram gated sodium channel (NaVSp1) PD forms a stand-alone, ion selec- amounts required for extensive structural studies. α tive pore (NaVSp1p) that is tetrameric, -helical, and that forms Here, we show that a protein dissection approach produces functional, sodium-selective channels when reconstituted into lipid folded, electrophysiologically active pore domains (PDs) from BIOPHYSICS AND bilayers. Mutation of the Na Sp1p selectivity filter from LESWSM a set of bacterial Na s. These PDs are well-behaved, biochemi- V V COMPUTATIONAL BIOLOGY to LDDWSD, a change similar to that previously shown to alter ion cally tractable, stand-alone pores that can be produced in multi- selectivity of the bacterial sodium channel NaVBh1 (NaChBac), cre- milli-gram amounts. Application of a battery of biochemical ates a calcium-selective pore-only channel, CaVSp1p. We further and biophysical characterizations demonstrates that the PD-only show that production of PDs can be generalized by making proteins self-assemble as tetramers having α-helical structure and pore-only proteins from two other extremophile NaVs: one from form functional channels when incorporated into lipid bilayers. Alcanivorax borkumensis the hydrocarbon degrader (NaVAb1p), We further show that introduction of aspartic acid residues at and one from the arsenite oxidizer Alkalilimnicola ehrlichei key selectivity filter positions in the Silicibacter pomeroyi PD-only 1 1 (NaVAe1p). Together, our data establish a family of active pore-only channel NaVSp p creates a channel, CaVSp p, in which ion ion channels that should be excellent model systems for study selectivity is changed from sodium to calcium. Taken together, of the factors that govern both sodium and calcium selectivity our data demonstrate that the NaV pore-only proteins are active and permeability. Further, our findings suggest that similar dissec- and biochemically accessible ion channels. Because of their tion approaches may be applicable to a wide range of VGICs and, favorable biochemical properties, these NaV PDs should provide thus, serve as a means to simplify and accelerate biophysical, excellent model systems for the structural study of the factors that structural, and drug development efforts. govern sodium selectivity and permeability. Further, the dissec- tion strategy eliminates the potential complications that arise oltage-gated sodium channels (NaVs) are large polytopic from voltage-sensing domain (VSD) conformational heterogene- Vmembrane proteins involved in action potential generation ity and should be a generally applicable means to simplify studies in excitable cells and belong to an ion channel superfamily that of the PDs of other VGIC superfamily members. includes voltage-gated calcium channels (Ca s), voltage-gated V Results potassium channels (KVs), and transient receptor potential (TRP) channels (1, 2). Within the voltage-gated ion channel Creation of a Pore-Only NaV. We previously identified an NaV from – the marine sulfur-reducing bacterium S. pomeroyi (Na Sp1), (VGIC) superfamily, NaVs and CaVs are close relatives (1 3) that V which is functional when expressed in mammalian cells (8), as a share a topology of 24 transmembrane segments organized in −1 four homologous six-transmembrane repeats. These two families protein that could be highly expressed (approximately 20 mg L ) are also thought to share some common structure in the ion se- in Escherichia coli membranes (15). Despite the high expression lectivity filter despite having markedly different ion permeation level, size-exclusion chromatography analysis of purified, deter- 1 properties (4). Both are central to human neuromuscular, cardi- gent-solubilized NaVSp revealed a broad elution profile indica- ovascular, and neural physiology. Consequently, they are targets tive of a polydisperse sample that was not well suited to further for a host of pharmaceuticals used to treat a diverse set of – disorders and remain active targets for drug development (5 7). Author contributions: D.S., M.K., and D.L.M. designed research; D.S., M.K., R.A.R., Recently, single subunit, six-transmembrane segment NaVs have S.W., and J.H. performed research; D.S., M.K., R.A.R., S.W., and J.H. contributed new been identified in a large number of bacteria from diverse envir- reagents/analytic tools; D.S., M.K., R.A.R., S.W., J.H., A.B., and D.L.M. analyzed data; onments (8, 9). These channels show clear similarities to eukar- and D.S., M.K., and D.L.M. wrote the paper. The authors declare no conflict of interest. yotic NaVs and CaVs (2, 9, 10), suggesting that the prokaryotic channels may have been ancestors of the more complex verte- This article is a PNAS Direct Submission. brate channels. Freely available online through the PNAS open access option. Despite the central importance of NaVs, nothing is known 1To whom correspondence should be addressed. E-mail: [email protected]. about the high-resolution structure of the NaV transmembrane This article contains supporting information online at www.pnas.org/lookup/suppl/ portions. Present knowledge is limited to low-resolution electron doi:10.1073/pnas.1106811108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1106811108 PNAS ∣ July 26, 2011 ∣ vol. 108 ∣ no. 30 ∣ 12313–12318 Downloaded by guest on September 26, 2021 following removal of the affinity tags and purification (Fig. 1 A 1 and C). These properties made NaVSp p very suitable for further biochemical and biophysical characterization. Biophysical Characterization Demonstrates That NaVSp1p Is Folded and Forms Tetramers. Structurally characterized VGIC members α 1 have high -helical content (16, 24, 31, 32). To examine NaVSp p secondary structure, we used circular dichroism (CD) and mea- sured spectra in two different detergents, n-dodecyl-β-D-malto- pyranoside (DDM) and n-decyl-β-D-maltopyranoside (DM). 1 In both, NaVSp p showed the characteristic hallmark double minima of helical proteins (Fig. 2A) (33). These spectral signa- tures are similar to those reported for purified, detergent-solubi- 1 lized full-length NaVBh (NaChBac) (12, 13), electric eel NaV (34), and the pore-only potassium channel KcsA (35, 36) and Fig. 1. Creation of a pore-only Na .(A) Superdex 200 size-exclusion chroma- 1 α V indicate that NaVSp p has the high -helical content character- 1 tography of NaVSp in 200 mM NaCl, 0.3 mM DDM, 20 mM HEPES, pH 8.0. istic of the VGIC superfamily. Thermal denaturation experi- (Inset) Fifteen percent SDS-PAGE of the peak fraction. (B) Cartoon depicting ments monitored by CD indicate that Na Sp1p undergoes a the strategy for creating a pore-only channel. Two of the four VSDs are V shown along with the S4–S5 linker. Scissors indicate the dissection point cooperative loss of secondary structure (Fig. 2B) that is charac- (Left). (C) Superdex 200 size-exclusion chromatography of Na Sp1pin teristic of a folded protein and that resembles that seen in the full- V 1 200 mM NaCl, 0.3 mM DDM, 20 mM Hepes pH 8.0. (Inset) Fifteen percent length electric eel NaV (34). Moreover, NaVSp p has different SDS-PAGE of the peak fraction. degrees of stability in DDM and DM (apparent melting tempera- tures, Tms, of 44 °C and 52 °C, respectively) and the thermal tran- characterization (Fig. 1A). Thus, we sought alternative strategies sition in DDM can be made largely reversible (90% recovery of to produce biochemically tractable NaV samples. the signal at 222 nm) by the inclusion of 8% glycerol (Fig. S1B). Three lines of evidence have suggested that VGIC members Together, these spectral and thermal denaturation properties are composed of two domains that can fold independently, the 1 α suggest that
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