Dynamics transitions of the KvAP voltage sensor during lipid-dependent gating

In response to changes in membrane potential, the S4 helix of VSD undergoes a structural transition from resting/‘Down’ to activated/‘Up’ conformation, which is an extremely important step for the functioning of a voltage-gated ion channel. It is well known that the presence of phospholipid headgroups are an absolute requirement for the normal functioning of Kv channels whereas channels reconstituted in lipids lacking the phosphate headgroups renders the channel nonfunctional. Interestingly, apart from voltage-dependent gating, the gating of KvAP can also be influenced by the change in membrane lipid composition in a voltage-independent manner (i.e., lipid-dependent gating). Here, we have performed differential lipid reconstitution using liposomes composed of phospholipids (POPC/POPG) to stabilize the activated/‘Up’ conformation and using liposomes with a mixture of phospholipids and non-phospholipids (DOPC/cholesterol) to trap the isolated KvAP-VSD in its ‘Down’ conformation. The results, using site-directed NBD fluorescence on the entire S4 helix, show that the moving part of the KvAP sensor adopts a relaxed dynamics in its ‘Down’ state whereas a restricted dynamics is observed in its ‘Up’ state. Further, a variation in hydration dynamics is observed when the S4 transitions between the two functional states, which not only reduces the relative discrete population of S4 conformations, but also alters the lifetime distribution peaks of the S4 in ‘Down’ conformation. This strongly suggests that the structural dynamics and conformational heterogeneity of the S4 is significantly altered which correlates with the change in conformation of the sensor during gating in membranes. Importantly, our ‘Down’ state results strongly indicate a bending/breakage from the middle of the otherwise straight S4-helix in ‘Up’ conformation. Overall, our results highlight the impact of altered lipid-protein interface that has a strong influence on the structural dynamics of the functionally-important conformations of the KvAP sensor.