Pless SA , Elstone FD , Niciforovic AP , Galpin JD , Yang R , Kurata HT , Ahern CA .

GM087519 NIGMS NIH HHS , MOP-56858 Canadian Institutes of Health Research , MOP-97998 Canadian Institutes of Health Research , R01 GM106569 NIGMS NIH HHS , R01GM106569 NIGMS NIH HHS , U54 GM087519 NIGMS NIH HHS , MOP-56858 CIHR, MOP-97998 CIHR

	Figure 1. Highly conserved acidic and aromatic side chains in the VSDs of voltage-gated ion channels. (A) Sequence alignment of S1, S2, and S3 segments of different voltage-gated ion channels: Nav1.4, NavAb, and Shaker potassium channels. The S2 HC is highlighted in green; negatively charged side chains in the ENC and the INC are highlighted in red. (B) Structure of the NavAb VSD (Protein Data Bank accession no. 3RVY; Payandeh et al., 2011). The conserved S4 arginines, as well as conserved acidic and aromatic side chains in S2 and S3, are shown in stick representation (note that S1 was omitted for clarity).
	Figure 2. ENC electrostatic contributions are critical in DI and DII only. (A and D) Sample traces for currents recorded for WT and mutants at the S1 and the S2 ENC in DI–DIV. (B, C, E, and F) G-V (B and E) and SSI curves (C and F) for WT and mutants at the S1 and the S2 ENC; the insets in C and F show a bar graph representing the average time constants for fast inactivation (τ) for a depolarizing voltage step to −15 mV for WT and the mutants; *, statistical difference to WT values in an unpaired t test (P < 0.01). Note that Asn1389 in the S2 ENC in DIV had been mutated to both acidic and basic side chains previously with no functional consequence and was thus not studied further here (Groome and Winston, 2013). Bars: horizontal, 5 ms; vertical, 200 nA. Voltage steps were from −40 to +20 mV in 10-mV increments. Insets show energy-minimized structures and ESP maps of side chains (red, −100 kcal/mol; green, 0 kcal/mol; blue, +100 kcal/mol; see Pless et al., 2011b, for details).
	Figure 3. Removing the negative charge in the INC has little effect on channel activation. (A and B) G-V (A) and SSI curves (B) for WT and mutants in which the S2 INC was neutralized through introduction of Nha or Gln (DI, Glu171TAG + Nha; DII, Glu624Gln; DIII, Glu1079Gln; DIV, Glu1399TAG + Nha). (C and D) G-V (C) and SSI curves (D) for WT and mutants in which the S3 INC was neutralized through the introduction of Asn (DI, Asp197Asn; DII, Asp646Asn; DIII, Asp1101Asn; DIV, Asp1420Asn). The insets in B and D show bar graphs representing the average time constants for fast inactivation (τ) for a depolarizing voltage step to −15 mV. *, statistical difference to WT values in an unpaired t test (P < 0.01).
	Figure 4. Replacing the S2 aromatic with Leu has drastic functional consequences in DII–DIV. (A) Chemical structure of Phe and Leu. (B) Sample traces for currents recorded from WT or mutants that replaced the S2 aromatic with Leu (DI, Tyr168Leu; DII, Phe621Leu; DIII, Phe1076Leu; DIV, Phe1396Leu). Bars: horizontal, 5 ms; vertical, 500 nA. Voltage steps were from −40 to +20 mV in 10-mV increments. (C and D) G-V (C) and SSI curves (D) for WT and mutants in which the S2 aromatic was replaced by Leu; the inset in D shows a bar graph representing the average time constants for fast inactivation (τ) for a depolarizing voltage step to −15 mV for WT and the Leu mutants in DI–DIV. *, statistical difference to WT values in an unpaired t test (P < 0.01).
	Figure 5. Removing the negative ESP of the S2 aromatic has minimal functional consequences. (A) Chemical structures and ESP maps of Phe and 3,4,5-trifluoro-Phe (F3-Phe) (ESP: red, −25 kcal/mol; green, 0 kcal/mol; blue, +25 kcal/mol; see Pless et al., 2011a, for details). (B) Sample traces for currents recorded from WT or mutants in which F3-Phe has been introduced in the S2 HC (DI, Tyr168TAG + F3-Phe; DII, Phe621TAG + F3-Phe; DIII, Phe1076TAG + F3-Phe; DIV, Phe1396TAG + F3-Phe). Bars: horizontal, 5 ms; vertical, 500 nA. Voltage steps were from −40 to +20 mV in 10-mV increments. (C and D) G-V (C) and SSI curves (D) for WT and mutants in which the S2 aromatic was replaced by F3-Phe; the inset in D shows a bar graph representing the average time constants for fast inactivation (τ) for a depolarizing voltage step to −15 mV for WT and the F3-Phe mutants in DI–DIV.
	Figure 6. Introduction of a Trp highlights functional differences to potassium channel VSDs. (A) Chemical structure of Phe and Trp. (B) Sample traces for currents recorded from WT or mutants in which Trp has been introduced in the S2 HC (DI, Tyr168Trp; DII, Phe621Trp; DIII, Phe1076Trp; DIV, Phe1396Trp). The inset below DI Trp shows normalized currents recorded from WT (black) and the DI Trp mutant (red) in response to a depolarization to +15 mV. Bars: horizontal, 5 ms; vertical, 500 nA. Voltage steps were from −40 to +20 mV in 10-mV increments. (C and D) G-V (C) and SSI curves (D) for WT and mutants in which the S2 aromatic was replaced by Trp; the inset in D shows a bar graph representing the average time constants for fast inactivation (τ) for a depolarizing voltage step to −15 mV for WT and the Trp mutants in DI–DIV. *, statistical difference to WT values in an unpaired t test (P < 0.01). Note that the corresponding value for Tyr168Trp could not be determined because of the lack of significant ionic current at −15 mV.
	Figure 7. The disrupted inactivation phenotype of Tyr168Trp is caused by the Trp H-bonding ability. (A) Sample traces for currents recorded from WT and mutants that replaced the DI S2 Tyr with Trp or Ind, respectively (chemical structures are shown next to the current traces). Bars: horizontal, 5 ms; vertical, 500 nA. Voltage steps were from −40 to +20 mV in 10-mV increments. (B and C) G-V (B) and SSI curves (C) for WT and mutants in which the DI S2 Tyr was replaced with Trp or Ind.

Arrigoni, The voltage-sensing domain of a phosphatase gates the pore of a potassium channel. 2013, Pubmed, Xenbase

Arrigoni, The voltage-sensing domain of a phosphatase gates the pore of a potassium channel. 2013, Pubmed , Xenbase
Bezanilla, How membrane proteins sense voltage. 2008, Pubmed
Bosmans, Deconstructing voltage sensor function and pharmacology in sodium channels. 2008, Pubmed , Xenbase
Burley, Amino-aromatic interactions in proteins. 1986, Pubmed
Capes, Gating transitions in the selectivity filter region of a sodium channel are coupled to the domain IV voltage sensor. 2012, Pubmed
Cashin, Chemical-scale studies on the role of a conserved aspartate in preorganizing the agonist binding site of the nicotinic acetylcholine receptor. 2007, Pubmed , Xenbase
Catterall, Ion channel voltage sensors: structure, function, and pathophysiology. 2010, Pubmed
Cha, Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation. 1999, Pubmed , Xenbase
Chanda, Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation. 2002, Pubmed , Xenbase
Cheng, Functional interactions of voltage sensor charges with an S2 hydrophobic plug in hERG channels. 2013, Pubmed , Xenbase
DeCaen, Gating charge interactions with the S1 segment during activation of a Na+ channel voltage sensor. 2011, Pubmed
DeCaen, Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation. 2008, Pubmed
DeCaen, Sequential formation of ion pairs during activation of a sodium channel voltage sensor. 2009, Pubmed
Delemotte, Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations. 2011, Pubmed
Dougherty, Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. 1996, Pubmed
Gallivan, Cation-pi interactions in structural biology. 1999, Pubmed
Goldschen-Ohm, Multiple pore conformations driven by asynchronous movements of voltage sensors in a eukaryotic sodium channel. 2013, Pubmed , Xenbase
Groenewegen, A cardiac sodium channel mutation cosegregates with a rare connexin40 genotype in familial atrial standstill. 2003, Pubmed , Xenbase
Groome, S1-S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation. 2013, Pubmed
Klassen, Non-linear intramolecular interactions and voltage sensitivity of a KV1 family potassium channel from Polyorchis penicillatus (Eschscholtz 1829). 2008, Pubmed , Xenbase
Lacroix, Control of a final gating charge transition by a hydrophobic residue in the S2 segment of a K+ channel voltage sensor. 2011, Pubmed , Xenbase
Lacroix, Intermediate state trapping of a voltage sensor. 2012, Pubmed , Xenbase
Lin, R1 in the Shaker S4 occupies the gating charge transfer center in the resting state. 2011, Pubmed , Xenbase
Long, Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. 2005, Pubmed
Lu, Ion conduction pore is conserved among potassium channels. 2001, Pubmed
McCusker, Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing. 2012, Pubmed
McNair, SCN5A mutation associated with dilated cardiomyopathy, conduction disorder, and arrhythmia. 2004, Pubmed
McPhee, A critical role for the S4-S5 intracellular loop in domain IV of the sodium channel alpha-subunit in fast inactivation. 1998, Pubmed , Xenbase
McPhee, A mutation in segment IVS6 disrupts fast inactivation of sodium channels. 1994, Pubmed , Xenbase
Muroi, Molecular determinants of coupling between the domain III voltage sensor and pore of a sodium channel. 2010, Pubmed
Nowak, In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system. 1998, Pubmed , Xenbase
Paldi, Coupling between residues on S4 and S1 defines the voltage-sensor resting conformation in NaChBac. 2010, Pubmed
Papazian, Electrostatic interactions of S4 voltage sensor in Shaker K+ channel. 1995, Pubmed , Xenbase
Payandeh, Crystal structure of a voltage-gated sodium channel in two potentially inactivated states. 2012, Pubmed
Payandeh, The crystal structure of a voltage-gated sodium channel. 2011, Pubmed
Planells-Cases, Mutation of conserved negatively charged residues in the S2 and S3 transmembrane segments of a mammalian K+ channel selectively modulates channel gating. 1995, Pubmed , Xenbase
Pless, Unnatural amino acids as probes of ligand-receptor interactions and their conformational consequences. 2013, Pubmed
Pless, Contributions of counter-charge in a potassium channel voltage-sensor domain. 2011, Pubmed , Xenbase
Pless, Molecular basis for class Ib anti-arrhythmic inhibition of cardiac sodium channels. 2011, Pubmed
Pless, Contributions of conserved residues at the gating interface of glycine receptors. 2011, Pubmed
Santarelli, A cation-pi interaction discriminates among sodium channels that are either sensitive or resistant to tetrodotoxin block. 2007, Pubmed , Xenbase
Sato, Molecular dissection of the contribution of negatively and positively charged residues in S2, S3, and S4 to the final membrane topology of the voltage sensor in the K+ channel, KAT1. 2003, Pubmed , Xenbase
Sato, Integration of Shaker-type K+ channel, KAT1, into the endoplasmic reticulum membrane: synergistic insertion of voltage-sensing segments, S3-S4, and independent insertion of pore-forming segments, S5-P-S6. 2002, Pubmed , Xenbase
Schwaiger, The conserved phenylalanine in the K+ channel voltage-sensor domain creates a barrier with unidirectional effects. 2013, Pubmed , Xenbase
Schwaiger, 3₁₀-helix conformation facilitates the transition of a voltage sensor S4 segment toward the down state. 2011, Pubmed
Seoh, Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. 1996, Pubmed , Xenbase
Sheets, The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4. 1999, Pubmed
Tao, A gating charge transfer center in voltage sensors. 2010, Pubmed , Xenbase
Tiwari-Woodruff, Electrostatic interactions between transmembrane segments mediate folding of Shaker K+ channel subunits. 1997, Pubmed , Xenbase
Tiwari-Woodruff, Voltage-dependent structural interactions in the Shaker K(+) channel. 2000, Pubmed , Xenbase
Ulbricht, Sodium channel inactivation: molecular determinants and modulation. 2005, Pubmed
Watanabe, Striking In vivo phenotype of a disease-associated human SCN5A mutation producing minimal changes in vitro. 2011, Pubmed
West, A cluster of hydrophobic amino acid residues required for fast Na(+)-channel inactivation. 1992, Pubmed , Xenbase
Wu, State-dependent electrostatic interactions of S4 arginines with E1 in S2 during Kv7.1 activation. 2010, Pubmed , Xenbase
Yang, Evidence for voltage-dependent S4 movement in sodium channels. 1995, Pubmed
Yarov-Yarovoy, Structural basis for gating charge movement in the voltage sensor of a sodium channel. 2012, Pubmed
Zhang, Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel. 2012, Pubmed
Zhang, Contribution of hydrophobic and electrostatic interactions to the membrane integration of the Shaker K+ channel voltage sensor domain. 2007, Pubmed
Zhong, From ab initio quantum mechanics to molecular neurobiology: a cation-pi binding site in the nicotinic receptor. 1998, Pubmed