| Popis: |
The molecular correlates of the large energy barriers, underlying the strong temperature dependent modulation of activation kinetics of thermp-TRP channels, are virtually unknown. Recent evidence, based on de novo design of a temperature-modulated channel, has suggested a general mechanism of temperature dependent gating - conformational state dependent changes in hydration of critical residues alters the specific heat capacity change of gating thereby rendering the gating sensitive to temperature. Here, we study whether such a hydration-inspired hypothesis can account for effect of temperature on gating kinetics. Our model system was the Shaker KV channel wherein we focused our attention on the four principle gating-charge residues on the S4 segment of the channel. We find that neutralization of the fourth gating charge to alanine results in a 200-fold deceleration of channel deactivation kinetics as the temperature is reduced by 20C, although its apparent open-probability vs voltage curves, at the two temperatures, are virtually superimposable. By neutralizing R371 to amino acids of varying polarities, we find that while temperature has no effect on the open-probability vs voltage relationships, the deactivation kinetics is affected by temperature in a polarity correlated fashion i.e. hydrophobic perturbations produce a strong temperature dependence but polar perturbations result in modest, wild-type like, effects. These results are explained by a model in which the last step of voltage-dependent activation of the channel involves translocation of the fourth charge site (371) from the hydrated internal crevice to the hydrated external crevice through a hydrophobic septum, during which the charge is transiently dehydrated. These results provide a molecular framework to rationalize the effect of temperature on gating kinetics and also describe the unique role of hydration in governing the energy landscape of gating. |
