Pless SA et al. (2014), Asymmetric functional contributions of acidic a...

XB-ART-50484

J Gen Physiol 2014 May 01;1435:645-56. doi: 10.1085/jgp.201311036.

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Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains.

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

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Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (NaV1.4). The results show that the highly conserved aromatic side chain constituting the S2 HC makes distinct functional contributions in each of the four NaV domains. No obvious cation-pi interaction exists with nearby S4 charges in any domain, and natural and unnatural mutations at these aromatic sites produce functional phenotypes that are different from those observed previously in Kv VSDs. In contrast, and similar to results obtained with Kv channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect on the voltage dependence of activation in any of the four domains. Interestingly, countercharge was found to play an important functional role in the ENC of DI and DII, but not DIII and DIV. These results suggest that electrostatic interactions with S4 gating charges are unlikely in the INC and only relevant in the ENC of DI and DII. Collectively, our data highlight domain-specific functional contributions of highly conserved side chains in NaV VSDs.

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Species referenced: Xenopus
Genes referenced: cntn1 nav1 scn4a tbx2

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	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.

References [+] :

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