The Nature of the Energy Barrier for the Charge Movement in Voltage-Sensors

Abstract

Voltage-sensors (VS) are protein domains which transduce changes in membrane potential into conformational changes thereby controlling the opening of a pore or the activity of a phosphatase domain. This operation creates measurable gating currents (Ig) by a local rearrangement of charged groups in the VS. Although there is high conservation of these gating charges among VS, the kinetics of Ig varies greatly, spreading over several orders of magnitudes. These kinetic differences explain the temporal separation of Na+ and K+ conductances in excitable cells, critical for the generation and propagation of the action potential. Despite its fundamental importance, the molecular mechanism underlying this phenomenon is not well understood. Here, we identified in the Shaker K+ channel two hydrophobic residues Ile241 and Ile287 located in S1 and S2 respectively which directly interact with S4 arginines during gating charge movement. We show that Shaker gating kinetics can be speeded up ∼ 3 fold by mutating Ile287 to more hydrophilic residues threonine or serine. Interestingly, voltage-dependent sodium channel (Nav) possesses threonines in domains I-III at positions homologous to Ile287 and exhibit Ig ∼ 10 times faster than that of Shaker. Mutating these threonines to isoleucines in Nav1.4 slows down Ig 2-3 times. Moreover, the sensing currents of the voltage-sensitive phosphatase Ci-VSP, which are ∼ 8 times slower than that of Shaker, can be speeded 3-4 fold by substituting Ile126, (homologous to Ile241 in Shaker) to threonine. Taken together, our data indicate that the side chains of these two key positions form a hydrophobic plug separating the external and internal media. This plug constitutes the main energy barrier for charge crossing and its amplitude is modulated by its degree of hydrophobicity, thus controlling the kinetics of the sensor. Support: NIH-GM030376.