Abstract
DOTAP is a bilayer-forming lipid containing a positively charged trimethyl-ammonium group instead of a negatively charged phosphodiester. When reconstituted in pure DOTAP membranes, KvAP is unable to open due to large positive voltage shifts in its mid-point of activation. Based on these results, it has been hypothesized that non-phosphate lipids stabilize the down (resting) conformation of the voltage sensor. We have tested this idea by studying the influence of non-phospholipids on the gating properties of channels with inverse electromechanical coupling. In contrast to most Kv channels, the pore domain of hyperpolarization-activated channels opens with the downward movement of S4 (at hyperpolarizing potentials). Therefore, for any hyperpolarization-activated channel, reconstitution in DOTAP should stabilize the down state of the VSD, favoring the open state and not the closed state. We carried out a CW-EPR analysis of the local structure and dynamics of the prokaryotic hyperpolarization activated potassium channel MVP, after reconstitution in either PC:PG (3:1) or DOTAP liposomes, in the absence of a resting potential. We focused on residues in the S4, S4-S5 linker, and lower S6 of MVP as a way to monitor the key regions that participate in voltage sensing and gate coupling. In PC:PG phospholipids, the inner bundle gate of MVP is closed, as seen from strong spin-spin coupling between residues lining the S6. In contrast, our findings suggest that DOTAP biases the conformation of the VSD in MVP towards the down state, which leads to a structural reorganization of the lower S5 region and conformational changes in the pore domain. The activation gate displays key hallmarks associated with the open conformation. These lipid-driven structural changes point to a working model for the inverse electromechanical coupling in hyperpolarization-activated channels.
Published Version
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