Towards a physical understanding of thermodynamic and kinetic properties of gating in voltage-dependent ion channels

Thermodynamic and kinetic properties of the voltage-sensing mechanism of the non-inactivating Shaker H4 potassium channel were inferred from voltage clamp experiments. The findings from these studies lead to a consistent view of the activation pathway of the Shaker channel. Non-stationary fluctuation analysis of gating currents revealed two stages of channel activation. The first stage consists of numerous transitions which individually carry little charge, generating small fluctuations. The second stage contains at least one dominant transition which moves about 2.4 elementary charges. Temperature studies of ionic and gating currents suggest that entropy is lowered during the early transitions, then there an increase in entropy with large enthalpic barriers in the second stage, followed by a decrease in entropy with the final voltage-insensitive opening step. A theoretical relation was developed between the charge intimately responsible for channel opening (activation charge) and the total gating charge. It was shown that if the two quantities match at every voltage, then the entire gating charge movement is energetically coupled to channel opening. This was tested experimentally on Shaker and the hypothesis was confirmed. In addition, point mutations which neutralize the charge of putative voltage sensing residues in the second and fourth transmembrane domains decreased total activation charge displacement by up to one half. Involvement of at least two transmembrane domains in the gating process implies that gross macroscopic changes of the channel protein occur during activation. Considering the fact that the channel lives in a solvent environment, then conformational changes of large magnitude are best modeled by a diffusion process. Numerical and Monte Carlo techniques were developed to solve the Smoluchowski diffusion equation for an arbitrary landscape. It was shown that for landscapes containing large barriers, there is a separation of time scale in the gating current. The diffusional time scale ( m s) features the early drift event of gating, which has been observed experimentally in high bandwidth recordings. The physiological time scale (ms) features the regular slow components of gating that are well modeled with a discrete state Markov (DSM) scheme.