The resting and activated conformations of the voltage sensor of Ci-VSP from functional and solvent accessibility determinations

ORCiD

Carlos A. Villalba-Galea: 0000-0002-6489-4651

Document Type

Conference Presentation

Conference Title/Conference Publication

Biophysical Journal

Organization

Biophysical Society 56th Annual Meeting

Location

San Diego, CA

Conference Dates

February 25-29, 2012

Date of Presentation

2-25-2012

ISSN

0006-3495

Volume

102

Issue

3, Supplement 1

DOI

10.1016/j.bpj.2011.11.224

First Page

36a

Abstract

The voltage sensor domain (VSD) is responsible for electromechanical transduction in voltage-gated ion channels and enzymes. In all known VSDs, both architecture and voltage-sensing mechanism are conserved: the positive charged residues (R/K) on the fourth transmembrane segment S4 respond to the voltage change across the membrane, which trigger its own conformation change leading to the response of downstream domain. A wealth of biophysical information on voltage sensors in the last two decades has revealed one of the major functional states - “up” or activated state. However, the structure and functional properties of the “down” or resting state remains controversial. Here, we show electrophysiological and structural studies of the voltage sensor from Ciona intestinalis voltage sensitive phosphatase (Ci-VSP), that point to conformational transitions between the resting and activated conformations of the sensor. The voltage dependence of Ci-VSP mutants, analyzed by gating charge measurement in oocytes, show significant shift in their Q-V relationships along the voltage axis (R217E −60 mV, R217Q −20 mV, WT +60 mV, D136N +130 mV). At 0 mV, these mutants populate different functional states under biochemical conditions: WT and D136N mostly in the “down” state while R217E is mostly in the “up” state. A Ci-VSD biochemical preparation was developed for each of the four mutants and studied by site-directed spin labeling EPR (SDSL-EPR) methods in proteoliposomes. Mobility and accessibility information revealed the secondary structure of transmembrane segments and their positions relative to membrane and each other, suggesting the extend and direction of the motion of S4 between “up” and “down” states. These results are consistent with the down movement of S4 under hyperpolarization and render critical structural information, that allow us to propose a gating mechanism for Ci-VSD.

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