Functional Analysis of Kv1.2 and Paddle Chimera Kv Channels in Planar Lipid Bilayers

Abstract

SummaryVoltage-dependent K + channels play key roles in shaping electrical signaling in both excitable as well as non-excitable cells. These channels open and close in response to the voltage changes across the cell membrane. Many studies have been carried out in order to understand the voltage sensing mechanism. Our laboratory recently determined the atomic structures of a mammalian voltagedependent K + channel Kv1.2 and a mutant of Kv1.2 named the 'paddle-chimera' channel, in which the voltage sensor paddle was transferred from Kv2.1 to Kv1.2. These two structures provide atomic descriptions of voltage-dependent channels with unprecedented clarity. Until now the functional integrity of these two channels biosynthesized in yeast cells have not been assessed. Here we report the electrophysiological and pharmacological properties of Kv1.2 and the paddle chimera channels in planar lipid bilayers. We demonstrate that Pichia yeast produce 'normally functioning' mammalian voltage-dependent K + channels with qualitatively similar features to the Shaker K + channel in the absence of the N-terminal inactivation gate, and that the paddle chimera mutant channel functions as well as Kv1.2. We find, however, that in several respects the Kv1.2 channel exhibits functional properties that are distinct from Kv1.2 channels reported in the literature.

RETRACTED: Voltage sensor ring in a native structure of a membrane-embedded potassium channel

“…However, the positions and orientations of the S4 arginine residues in these structures vary substantially (15)(16)(17)(18)(19). Electrophysiological studies have shown that membrane-embedded voltage-gated potassium channel from Aeropyrum pernix (KvAP), Kv2.1, and voltage-gated sodium channel from Aerobacter butzleri (NavAb) held at 0 mV become inactivated over time (17,22,24). Therefore, the X-ray structures of these three channels, if completely physiological, should all reflect the inactivated states (state I), not necessarily the states to which they were assigned.…”

Voltage-dependent K + channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane

“…Paddle chimera protein (␣ and ␤1 subunits) was expressed, purified and reconstituted as described in ref. 15.…”

“…1 actually represent the very same channels functioning in different membranes. The Kv channel is called ''paddle chimera,'' a Kv1.2 channel with a voltage sensor paddle from Kv2.1 (14,15). The properties of rapid activation, inactivation, a negative midpoint voltage and sensitivity to VSTx1 are observed when the channels are reconstituted into planar bilayers consisting of the lipids 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-glycero-3-phospho-1-glycerol (POPG) in a 3:1 ratio.…”

“…Channels studied in planar bilayers first have to be synthesized in yeast cells and then purified in detergent, reconstituted into lipid vesicles and fused with the planar bilayer (15). Channels studied in oocyte membranes are synthesized by the oocytes after injection of RNA.…”

Single channel and ensemble hERG conductance measured in droplet bilayers

“…However, our previous measurements of hERG did not result in any observations of the expected temporal or voltage dependent conductance of ion channel ensembles,{Portonovo et al 2013#34} similar to others' measurements, {Zhang et al 2012 #38} although Oshima and coworkers reported voltage dependent single channel conductance. {Oshima et al 2013 #33} Temporal and voltage dependence of conductance for purified ion channels reconstituted in lipid bilayers has been previously reported by Tao and MacKinnon (Tao and MacKinnon 2008) and Schmidt and MacKinnon (Schmidt et al 2009) for K v 1.2 and K v AP channels, respectively. In the work described here, we used droplet bilayers to measure ion channel currents from membrane preparations of hERG-expressing cells at the single channel and ensemble levels, demonstrating ion selectivity and dose-dependent inhibition by E-4031 and dofetilide, with IC 50 values of 17 nM and 9.65 μM.…”