Neuronal calcium channels: Permeation and channel agonists
Description
All cells have proteins to transport calcium ions across the membrane. Voltage-dependent calcium channels comprise one class, and have a charged domain responding to changes in transmembrane voltage to regulate channel opening. This study investigates the two fundamental properties of these channels: permeation and gating. Permeation studies ion movement through open channels to determine the mechanism of ion selectivity and high ion flux. Gating properties define the conditions for channel opening and closing Calcium ions flowing through calcium channels bind to certain sites in the channel pore, which prevents other ions from permeating. Binding site number and location with respect to the electrical field are unknown. By studying ion flow through calcium channels using electrophysiology and computer models, we determined: (1) Permeation is voltage-dependent, placing at least two binding sites within the electrical field; (2) This voltage dependence is best observed at voltages more depolarized than 0 mV, which explains why it was not previously recognized; (3) Existing two- and three-site models failed to reproduce our data over a range of voltages and concentrations. Our four-site model fit these data and reproduced known features of calcium channel permeation The N-channel gate closes rapidly at hyperpolarized voltages, limiting channel studies to positive voltages. An agonist for N-channel, similar to Bay-K8644 for L-channels, would abridge the problem by allowing longer channel openings and would be a novel pharmacological tool We found that roscovitine, a known kinase inhibitor, independently and stereospecifically slowed the closing of N-, P/Q- and R-type calcium channels by binding to their open state, thus establishing it as a unique agonist of the CaV2 family. Roscovitine also inhibited almost all channels tested at relatively high (≥100 muM) concentrations. This effect was slow and may have resulted from kinase inhibition. However, potassium channels were strongly inhibited (IC50 = 23 muM) by a mechanism involving open channel block. Since potassium channel inhibition is known to be antitumorigenic, we speculate that part or all of roscovitine's anticancer properties come from its block of open potassium channels. Finally, using roscovitine we confirmed predictions from our modeling of permeation