Mechanism of Voltage Gating in the Voltage-Sensing Phosphatase Ci-VSP

Abstract

The conformational changes in voltage-sensing domain (VSD) are driven by the transmembrane electric field acting on charges and countercharges. Yet, the overall energetics and detailed mechanism of this process are not fully understood. Here, we determined free energy and displacement charge landscapes, as well as major conformations corresponding to a complete functional gating cycle in the isolated voltage-sensing domain of the phosphatase Ci-VSP (Ci-VSD) comprising four transmembrane helices (segments S1-S4). Molecular dynamics simulations highlight the extent of S4 movements. In addition to the crystallographically determined activated Up and resting Down states, the simulations predict two novel Ci-VSD conformations: a deeper resting state (Down-minus) and an extended activated (Up-plus) state. These additional conformations were experimentally probed via systematic cysteine mutagenesis with metal-ion bridges and the engineering of proton conducting mutants at hyperpolarizing voltages. These results show that voltage activation involves sequentially populating these four states in a stepwise way, translating one arginine across the membrane electric field per step, transferring ~3 e0 charges.

State Dependent Anionic Pore Currents Conducted by Single Countercharge Mutants in a Voltage-Sensing Phosphatase

“…Particularly, an apparent positive shift of the Q-V curve for the D136A, D136N and D151N mutants was observed at low pH. WE have shown previously that adding of 100 μM Cd 2+ does not increase the net OFF charge Q in the V219C/D136A and V219C/D151A mutants of Ci-VSP (48), further confirms that neutralization of countercharge residues D136 and D151 cannot conduct omega currents.…”

“…Since the external buffer contains only large organic ions (NMDG + and CH 3 SO 3 - ), we first suspected that the currents were carried out by protons or hydroxides. If the mutant conducts protons, inward currents should increase at low pH, as is the case for omega currents in Ci-VSP gating charge mutants of ( 48 , 58 ). However, inward currents decreased at low pH.…”

“…In other words, the transition rate between adjacent conformational states increased at high pH. However, there was no change in WT Ci-VSP net translocated charge ( Q ), and the Q-V curve reached a plateau at pHo between 6.5, and 9.0 ( 48 ). We argue that under these conditions the D129A mutant does not reach the “up-plus” state of the VSD (fully activated), even at +160 mV ( 48 ).…”

“…However, countercharges located in the S1-S3 segments of VSDs, have been associated to limited conformational rearrangement during voltage gating (21,(61)(62)(63)(64). To understand the molecular mechanism of the state dependent and selective conduction of omega currents, we performed MD simulations of the D129A and R226A mutants in Ci-VSD, as both mutants conduct transient proton currents during deactivation (48).…”

“…The WT VSD of Ci-CatSper3 can even conduct divalent cations Ca 2+ , Ba 2+ and Sr 2+ (46). Although voltage-sensitive phosphatases can neither conduct ions nor permeate protons like proton channels, mutations of S4 gating charge residues in Ci-VSP have been shown to conduct protons as well (47,48). The importance of role of these cationic omega currents caused by missense mutations is highlighted in certain inherited channelopathies, e.g., hypokalemic periodic paralysis, normokalemic periodic paralysis and cardiac arrhythmias, and may also have been linked to other pathologies (35,39,40,43,44,(49)(50)(51)(52).…”