Infield DT et al. (2018), Replacing voltage sensor arginines with citrull...

XB-ART-56317

J Gen Physiol 2018 Jul 02;1507:1017-1024. doi: 10.1085/jgp.201812075.

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Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape.

Infield DT , Lee EEL , Galpin JD , Galles GD , Bezanilla F , Ahern CA .

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Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance-voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.

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Species referenced: Xenopus
Genes referenced: cit mt-tr trna
GO keywords: potassium channel complex

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	Figure 1. Crystal structure of the Kv 1.2/2.1 chimera suggests electrostatic accommodation of gating charges within the voltage sensing domain. Labels correspond to the residues at homologous positions in Shaker. Voltage-sensing arginine residues are shown in blue, conserved glutamate residues are shown in red, and hydrophobic residues, including the conserved phenylalanine in the hydrophobic plug (F290 in Shaker), are shown in green.
	Figure 2. Encoding of citrulline into Shaker. (A) Structural and electrostatic representations of the side chains of arginine, citrulline, and glutamine. (B) Currents generated from co-injection of cRNA encoding R1TAG, R3TAG, and R4TAG with CIT-tRNA compared with those injected with cRNA and full-length (pCA-ligated) tRNA in parallel. Lack of expressed current in the pCA condition for a given position is consistent with faithful encoding of citrulline. Error bars indicate SEM.
	Figure 3. Lack of effect on activation of encoding citrulline at R1 in Shaker. (A–C) Voltage-dependent ionic currents from oocytes expressing WT, R362CIT-, and R362Q-Shaker channels. Oocytes were held at −80 mV and subjected to 100-ms pulses in 10-mV steps, followed by 100-ms pulses to −50 mV (WT, Cit) or −40 mV (Q) before returning to the holding potential. (D) Normalized G-V values. Error bars represent ±SEM. At R1, a positive charge is apparently dispensable for activation, provided the shape of arginine is maintained.
	Figure 4. Effects on activation of encoding citrulline at R3 in Shaker. (A and B) Voltage-dependent ionic currents from oocytes expressing R368CIT- and R368Q-Shaker channels. Oocytes were held at −80 mV and subjected to 100-ms pulses in 10-mV steps, followed by 100-ms pulses to −40 mV (Cit) or −30 mV (Q) before returning to the holding potential. (C) Normalized G-V values. Error bars represent ±SEM. Note that the same WT data are used here and in Fig. 3 D. At R3, neutralization via citrulline resulted in a rightward shift and change in activation slope, suggesting that a positive charge at this position is necessary for normal activation. However, the effect on activation is modest compared with mutation to glutamine.
	Figure 5. Outsized electrostatic role for R4 in channel deactivation. (A–C) Normalized G-V curves from COVC of WT-, R371Q-, and R371Cit-Shaker. Oocytes were held at −80 mV, then prepulsed to −120 mV for 200 ms, before a variable pulse ranging from 80 mV to −120 mV, followed by a postpulse to −120 mV, before returning to the original holding potential. (D) Normalized G-V values. Error bars represent ±SEM. (E) A comparison of the activation rate (τ) between WT- and R371Cit-Shaker as derived from the same data used for the G-V curves. (F) Deactivation rate for WT- and R317Cit-Shaker. For these data, oocytes were held at −80 mV, prepulsed to −120 mV for 200 ms, then pulsed to 80 mV for 50 ms, followed by a variable pulse ranging from 80 mV to −120 mV for 350 ms, before returning to the original holding potential.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase