Thermodynamical and kinetic properties of amino terminal and S4-S5 loop HERG channel mutants under steady-state conditions

¹ Universidad de Oviedo
² University of Oviedo

^* To whom correspondence should be addressed. E-mail: fbarros{at}uniovi.es.

Submitted on July 5, 2007
Revised on July 31, 2007
Accepted on 28 December 2007

	Abstract

Gating kinetics and underlying thermodynamical properties of human ether-á-go-go (eag)-related gene (HERG) K⁺ channels expressed in Xenopus oocytes were studied using protocols able to yield true steady-state kinetic parameters. Channel mutants lacking the initial 16 residues of the amino terminus before the conserved eag/PAS region showed significant positive shifts in activation voltage dependence associated to a reduction of z_g values and a less negative Go, indicating a deletion-induced displacement of the equilibrium towards the closed state. Conversely, a negative shift and an increased Go, indicative of a closed state destabilization, were observed in channels lacking the amino terminal proximal domain. Furthermore, accelerated activation and deactivation kinetics were observed in these constructs when differences in driving force were considered, suggesting that the presence of distal and proximal amino terminal segments contributes in wild-type channels to specific chemical interactions that raise the energy barrier for activation. Steady-state characteristics of some single point mutants in the intracellular loop linking S4 and S5 helices revealed a striking parallelism between the effects of these mutations and those of the amino terminal modifications. Our data indicate that besides the recognized influence of the initial amino terminus region on HERG deactivation, this cytoplasmic region also affects activation behaviour. They also suggest that not only a slow movement of the voltage sensor itself, but also delaying its functional coupling to the activation gate by some cytoplasmic structures perhaps acting on the S4-S5 loop, may contribute to the atypically slow gating of HERG.

Key Words: HERG, cytoplasmic domains, gating, potassium channel, thermodynamics