Thermodynamic and kinetic properties of amino-terminal and S4-S5 loop HERG channel mutants under steady-state conditions.
Gating kinetics and underlying thermodynamic properties of human ether-a-go-go-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 with a reduction of z(g) values and a less negative DeltaG(o), indicating a deletion-induced displacement of the equilibrium toward the closed state. Conversely, a negative shift and an increased DeltaG(o), indicative of 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 in addition to the recognized influence of the initial amino-terminus region on HERG deactivation, this cytoplasmic region also affects activation behavior. The data 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 possibly acting on the S4-S5 loop may contribute to the atypically slow gating of HERG.
Alonso-Ron
Carlos
C
Departamento de Bioquímica y Biología Molecular, Edificio Santiago Gascón, Campus del Cristo, Universidad de Oviedo, E-33006 Oviedo, Asturias, Spain.
de la Peña
Pilar
P
Miranda
Pablo
P
Domínguez
Pedro
P
Barros
Francisco
F
D019943:Amino Acid Substitution; D000818:Animals; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D009865:Oocytes / Q000502:physiology*; D011188:Potassium / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014982:Xenopus laevis
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D011188:Potassium