Leaflet-specific effects of charged lipids on a voltage-gated potassium channel

Voltage-gated potassium (Kv) channels possess distinct voltage-sensor (VSD) and pore (PD) domains, making it challenging to study domain-specific lipid effects. Here, we examined the functional modification of a prototypical Kv channel, KvAP, by phosphatidylglycerol (POPG) and phosphatidylserine (POPS) in mono-component asymmetric bilayers using the contact bubble bilayer (CBB) method. In these membranes, specific chemical modifications were distinguished from non-specific electrostatic (surface potential) effects by using the channel's gating as an intrinsic probe. No specific effects were observed when charged lipids were in the outer leaflet. When present in the inner leaflet, POPS exerted only a single specific effect: the acceleration of activation kinetics. In contrast, inner-leaflet POPG induced multiple, profound modifications: it also accelerated activation kinetics, but additionally shifted the conductance-voltage (G-V) curve hyperpolarized, attenuated the G-V slope, and accelerated inactivation kinetics. This clear contrast allows a domain-specific interpretation: the shared acceleration of activation is attributed to a general kinetic modulation of the VSD, while POPG's unique effects-impaired electromechanical coupling (attenuated slope) and accelerated inactivation-are attributed to specific chemical interactions with the VSD-PD linker and the PD, respectively. These results reveal a multi-site mechanism of lipid modulation dictated by leaflet asymmetry and headgroup chemistry.