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

Abstract

Voltage-gated K þ (Kv) channels are tetrameric assemblies in which each modular subunit consists of a voltage sensor and a pore domain. KvLm, the voltage-gated K þ 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 FL 4 -like voltage dependence at depolarizing potentials, but all four sensors are required to keep the channel closed during membrane hyperpolarization.lipid bilayers | membrane proteins | voltage gated potassium channels | membrane reconstitution D epolarization activated, voltage-gated K þ -selective channels (Kv) comprise four identical subunits each contributing a complete voltage sensor (S1-S4) and a quarter of the ion-conductive pore (S5-S6) (1, 2) (Fig. 1A). Upon membrane depolarization, the positively charged transmembrane segment S4 in each voltage sensor moves outward from its resting state and induces conformational changes in the pore. Between the resting and the open state, the channel undergoes a number of kinetic transitions. Transitions between the resting state and the last step that precedes opening and outward K þ flux define the voltage-dependent "activation" of the channel. In the Kv Shaker, activation entails at least five kinetic transitions observable as gating currents: Three early transitions that are voltage-dependent but noncooperative are followed by two late transitions (3-5). At the end of the activation pathway, each subunit is in an "activated-notopen" conformation (6, 7) referring to the state of the sensor and pore, respectively. Although it is well established that each sensor moves independently during the early transitions in activation (8), the nature of the interactions between subunits in Kv channels underlying the transition from activated-not-open to open (the opening transition) remains unsettled (6, 9-12). Structurally, the late kinetic transitions are considered to arise from conformational changes in the S4-S5 linker (7, 13), whereas the final opening transition (6, 7) entails a change in conformation of S6, which forms the bundle-crossing of the pore (activation gate).To determine the interplay between subunits underpinning the voltage dependence of the opening transition, it is necessary to uncouple it from the activation transitions that precede it and occur at similar rates. This dissection is possible when the opening transition is delayed or blocked. In Shaker, this has been achieved by blocking wit...

Grafting voltage and pharmacological sensitivity in potassium channels

“…In line with these experimental measurements, a recent all-atom molecular simulation suggested that full outward movement of S4 in one or two VSDs is insufficient to open the pore [1]. The experiment in the prokaryotic KvLm channels further revealed that three VSDs are required to promote classical voltage-dependent channel opening at depolarizing potentials [16]. In our study, either one or two VSDs are sufficient to open the channels in a voltage-dependent manner.…”

“…The work on KvLm channels shed new light on the roles of VSD. However, as it is a prokaryotic channel, the VSD of KvLm contains only three of the eight conserved charged residues known to be deterministic for voltage sensing in eukaryotic Kv channels, which may impede the generalization of these findings to eukaryotic Kv channels [15,16]. To the best of our knowledge, similar protein engineering strategies (physically remove VSDs) have not been successful in eukaryotic Kv channels.…”

“…Notably, the study on KvLm channels revealed that pore only module can populate at open state, suggesting that the pore of Kv channels may also reside in between closed-activated and open state [16]. The gating of the Kv channel pore involves both the lower bundle-crossing gate at the cytoplasmic entrance to the channel and the upper gate at the selectivity filter close to the outer mouth of the channel [54,55].…”

“…KvLm is a prokaryotic Kv channel from Listeria monocytogenes, characterized by high structural independence of the VSDs and the pore domain [15]. Taking advantage of this unique property, functional heteromers of the KvLm pore domain, assembled with one, two, or three VSDs, were produced by in vitro transcription and translation [16]. The results from this study suggested that three VSDs are sufficient to gate the channel, but all four sensors are required to close the channel [16].…”

“…Taking advantage of this unique property, functional heteromers of the KvLm pore domain, assembled with one, two, or three VSDs, were produced by in vitro transcription and translation [16]. The results from this study suggested that three VSDs are sufficient to gate the channel, but all four sensors are required to close the channel [16]. The work on KvLm channels shed new light on the roles of VSD.…”