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

Abstract

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 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.

Epilepsy-associated mutations in the voltage sensor of KCNQ3 affect voltage dependence of channel opening

“…However, co-injection of Shaker-R1UAG cRNA with Pyl-citrulline rendered robust voltage-activated potassium currents (Fig. S4, C and D), as previously reported (Infield et al, 2018a), thus validating the approach used here.…”

“…I introduced citrulline into KCNQ3-R230 channels using in vitro acylated tRNAs (Fig. 4 B), as previously reported (Infield et al, 2018a). Coinjection of citrulline-acylated pyrrolysine tRNA with cRNA of KCNQ3 channels bearing the amber UAG stop codon at 230 rendered voltage-activated potassium currents, which demonstrates successful encoding of citrulline (R230Cit; Fig.…”

Quantum Calculation of Proton and Other Charge Transfer Steps in Voltage Sensing in the Kv1.2 Channel

“…The R300cit mutation right shifts the Q-V curve. Given that R300 is part of the Y266,E183,R300 triad, it is not surprising that R300 behaves like Y266, and removing that path right shifts the curve, similarly to removing Y266; experimentally, it is somewhat larger, but not so much so as to make the interpretation implausible (75). Given that all other mutations and the WT calculations are consistent with the model presented here, the more extensive calculations are left for future work.…”

“…The second mutation, R303cit, in which a putative (in standard models) gating charge is replaced by the isosteric, but uncharged, citrulline side chain, showed a left shift (75). The energy for the proton transfer dropped in the mutant by approximately 10 kJ, equivalent to 4kBT, so that the calculation is consistent with a left shift, as in the experiment; the >50 kJ drop is the total for 0 mV, while the 10 kJ refers to the change in the barrier, that is, the energy at which the two curves in Fig.…”

Main-chain mutagenesis reveals intrahelical coupling in an ion channel voltage-sensor

“…In contrast to Q1, the midpoint voltage for movement of Q2 of V369Vah is shifted >40 mV in the depolarized direction (Supplementary Table 1 ), where it is well correlated with channel opening in this mutant. Removal of the hydrogen bond at 369 therefore results in perturbed function of the latter gating charges (presumably R3 and R4), which control channel activation 43 , 44 . Overall, the gating current data suggest the mechanism of the enhanced deactivation kinetics of V369Vah is due to an energetically unfavorable, and therefore an unstable, active S4 conformation.…”