6533b836fe1ef96bd12a09f0

RESEARCH PRODUCT

Two Structurally Distinct Pathways for the Voltage-Sensing S4 Helices

subject

description

In voltage-dependent ion channels, the movement of the voltage-sensing S4 helices produces gating currents. The charge displaced as a function of the membrane potential (Q-V) is well described by a sequential two-state Boltzmann relation, indicating that there are at least two steps of gating charge movement from their Resting state to the Active state. In addition, it has been shown that at a maintained positive potential, the S4 helices of voltage-gated Na, Ca and K channels and the voltage sensitive phosphatase Ci-VSP, undergo a slower secondary conformational transition stabilizing the sensor in a Relaxed (inactivated) state. From the Relaxed state, the Q-V relation exhibits a strong shift towards negative potentials when compared to the Q-V relation measured from the resting state. We engineered gating perturbations in the Shaker potassium channel, by substituting specific aromatic residues in positions spatially close to the S4. One of these mutants, in position I241 of S1, part of the hydrophobic plug of the voltage sensor, when mutated to tryptophan (I241W), produces a strong split in the Q-V when measured from the resting state. By labeling M356C with TMRM we also find the same split in the fluorescence-voltage curve. We propose that the presence of the tryptophan in the 241 position favors an interaction with one of the positively-charged arginines along the S4, thus stabilizing a fleeting intermediate state in the gating pathway. However, in the I241W mutant, the split in the Q-V almost disappears when the gating currents are measured from the relaxed state and the same result is seen with the fluorescence-voltage curve. This result and the effect of other tryptophan perturbations near the S4 segment strongly support the existence of two structurally distinct gating pathways for the movement of the S4 helices. Supported: NIHGM030376.