Functional and Crystallographic Studies of Hetero-Multimeric K+ Channels

Abstract

Voltage gated potassium channels (Kv channels) consist of four identical subunits that form a central permeation pathway in which changes in diameter of an inner constriction known as the activation gate (AG) catalyzes their transition from the closed to the open state. Kv channels play a major role in shaping and regulating the action potential in all excitable cells. Expressed as homotetramers as well as heterotetramers, Kv channels have a crucial role in excitability disorders such as arrhythmias, long QT syndrome, hyperactivity disorders, epilepsia and many other behavioral disorders. This renders heteromeric Kv channels as potential pharmaceutical targets but challenges arise because of their hetero-multimeric nature and diverse sequence identity, which make the smart design of safer and more specific therapeutic drugs difficult. Thus, it is of significant importance to study in detail the structure-function relationship in heteromeric Kv channels. Since KcsA is the bona fide structural and functional surrogate of the pore domain of Kv channels, we have constructed tandem-tetramer constructs of KcsA in which all four protomers are expressed as a single polypeptide chain by engineering linkers between the protomers. We were able to express, purify and solved the crystal structure of functional KcsA tandem-tetramer. This opens up a new and unprecedented avenue to introduce dose dependent mutations in the channel tetramer to evaluate the cooperative behavior of the channel function i.e., ion permeation, gating and ion selectivity, from a functional and structural point of view by using electrophysiology and X-ray crystallography approaches.Founding: AHA-11SDG5440003, NIH 1RO1GMo97159-01A1 and Welch Foundation BI-1757