Transitions near the open state in Shaker K(+)-channel: probing with temperature

Ionic currents from non-inactivating Shaker K(+)-channels were expressed in Xenopus oocytes and recorded with the cut-open oocyte voltage clamp technique. Kinetic properties of ionic currents were studied between 1.4 and 19 degrees C. The forward step of the last transition (...C --> O) of the activation pathway, just before the channel opening, was studied by the recovery of the ionic current after a short interval (< 500 microseconds) at a hyperpolarized potential (-90 mV) between two identical depolarizing pulses (0, 40 or 100 mV). Ionic currents under these experimental conditions are described by at least three kinetic components. The predominant kinetic component (corresponding to the ...C --> O transition) has a low temperature dependence (activation energy, Ea, around -5 kcal/mol) and it is voltage independent, suggesting that this step does not carry charge and has a mainly entropic energy barrier. The backward step (O --> C...) was studied from ionic tail currents at -120 mV after pulsing to 40 mV. The closure of the channel has a high temperature dependence (Q10 > 4), having an Ea of 24-25 kcal/mol. The closure of the channel also is voltage dependent, having a valence times fraction of the electric field, z beta of 0.5 eo. We conclude that the transition from the closed to the opened state has a net decrease in entropy and enthalpy, suggesting that the channel gets more ordered when it is open.