TEMPERATURE AND PRESSURE-DEPENDENCE OF SHAKER K-TYPE AND C-TYPE INACTIVATION( CHANNEL N)

Shaker-B potassium channels undergo rapid N-type and slow C-type inact ivation. While N-type inactivation is supposed to be mediated by occlu sion of the pore by the N-terminal protein structure, the molecular me chanisms leading to C-type inactivation are less well understood. Cons idering N-type inactivation as a model for a protein conformational tr ansition, we investigated inactivation of heterologously expressed Sha ker B potassium channels and mutants thereof, showing various degrees of C-type inactivation, under high hydrostatic (oil) pressure. In addi tion to the derived apparent activation and reaction volumes (Delta V) , experiments at various temperatures yielded estimates for enthalpic (Delta H) and entropic (T Delta S) contributions. N-type inactivation was accelerated by increasing temperature and slowed by high hydrostat ic pressure yielding at equilibrium Delta H = 76 kJ/mole, T Delta S = 82 kJ/mole, and Delta V = 0.18 nm(3) indicating that the transition to the N-type inactivated state is accompanied by an increase in volume and a decrease in order. N-terminally deleted Sh Delta 6-46 constructs with additional mutations at either position 449 or 463 were used to investigate C-type inactivation. In particular at high temperatures, i nactivation occurred in two phases indicating more than one process. A t equilibrium the following values were estimated for the major inacti vation component of mutant Sh Delta 6-46 T449A: Delta H = -64 kJ/mole, T Delta S = -60 kJ/mole, and Delta V = -0.25 nm(3), indicating that t he C-type inactivated state occupies a smaller volume and is more orde red than the noninactivated state. Thus, hydrostatic pressure affects N- and C-type inactivation in opposite ways.