Probing S4 Length Changes during Gating with LRET

Abstract

Voltage-activated proteins containing a Voltage Sensor Domain (VSD) respond to changes in the membrane potential by transferring across the electric field several positively charged residues (gating charges) located in the fourth transmembrane segment (S4). Even though the mechanistic details of gating charge translocation and S4 re-arrangement are presently unclear, a prevailing hypothesis suggests that the S4 segment adopts a 310 helix conformation during gating, thus aligning the gating charges and facilitating their transport across the membrane electric field. If a whole typical S4 segment were to change its conformation from an α-helix to a 310 helix, its length would stretch by about 8 Å. Here we tested the existence of such transition by measuring the length of the S4 segment during gating using the LRET technique. We used the VSD domain of the Ciona intestinalis Voltage-Sensitive Phosphatase (CiVSP), truncated from its phosphatase and phospholipid-binding domains, to genetically encode a lanthanide (Tb3+)-binding-tag at the extracellular end of S4 and the red fluorescent protein mCherry at its intracellular end. We expressed the protein in Xenopus oocytes from which we recorded gating currents using the cut-open and two-electrode voltage-clamp techniques. The distance-dependent efficiency of energy transfer between the two probes was measured at steady-state voltages and also during voltage pulse protocols of varying amplitudes and durations. Our technique could only detect distance changes larger than 2.5 Å. We found that in our CiVSP construct, the results are not consistent with the expected length change if the whole S4 were converted (and remained converted) from a 310 helix to an α-helix (or vice-versa) when the membrane potential is changed from −100 to +80 mV. Supported by NIH GM68044-07 and GM30376-30S1.