NMR study of the C-terminal domain gating and toxin binding in nanodisc-embedded potassium channels

The KcsA potassium channel is an excellent model for studying the structure and function of the important class of mammalian voltage-gated channels (Kv). Our research has a dual structural focus, the pH-dependent gating function of the cytoplasmic KcsA C-terminal domain (CTD), and the molecular basis for binding of toxin inhibitors to the pore region of the channel and their specificity.

Contrary to earlier studies, previous findings in our group using a peptide corresponding to KcsA residues 128–160 have indicated that the CTD acts as one of three independent pH-gates of the channel. Here we show for full-length KcsA solubilized in nanodiscs that CTD gating is pH dependent, shifting from the closed state at pH 7 to the open state at acidic pH as viewed by 1H,15N-HSQC and 1H,13C-HMQC spectra, the latter on a Leu/Val selectively methyl-protonated sample. These nuclear probes and EPR were applied to a constitutively TM2-open double mutant channel and observed a coupling between the TM2 and CTD gates.

Nanodisc-solubilized KcsA could also be used to study the binding of novel toxin inhibitors to KcsA. Upon binding to the channel, NMR detects toxin conformational changes that may explain the different binding pose exhibited by various toxins. Such molecular information is a critical step in the understanding of molecular factors that affect toxin-channel affinities, providing a structural view that can explain biochemical and electrophysiological results as well as guide drug-design efforts.