Tetrameric assembly of KvLm K+ channels with defined numbers of voltage sensors

Voltage-gated K⁺ (Kv) channels are tetrameric assemblies in which each modular subunit consists of a voltage sensor and a pore domain. KvLm, the voltage-gated K⁺ channel from Listeria monocytogenes, differs from other Kv channels in that its voltage sensor contains only three out of the eight charged residues previously implicated in voltage gating. Here, we ask how many sensors are required to produce a functional Kv channel by investigating heterotetramers comprising combinations of full-length KvLm (FL) and its sensorless pore module. KvLm heterotetramers were produced by cell-free expression, purified by electrophoresis, and shown to yield functional channels after reconstitution in droplet interface bilayers. We studied the properties of KvLm channels with zero, one, two, three, and four voltage sensors. Three sensors suffice to promote channel opening with FL₄-like voltage dependence at depolarizing potentials, but all four sensors are required to keep the channel closed during membrane hyperpolarization.