Extracellular Ca²⁺ Modulates the Effects of Protons on Gating and Conduction Properties of the T-type Ca²⁺ Channel α_1G (Ca_V3.1)

Since Ca²⁺ is a major competitor of protons for the modulation of high voltage–activated Ca²⁺ channels, we have studied the modulation by extracellular Ca²⁺ of the effects of proton on the T-type Ca²⁺ channel α_1G (Ca_V3.1) expressed in HEK293 cells. At 2 mM extracellular Ca²⁺ concentration, extracellular acidification in the pH range from 9.1 to 6.2 induced a positive shift of the activation curve and increased its slope factor. Both effects were significantly reduced if the concentration was increased to 20 mM or enhanced in the absence of Ca²⁺. Extracellular protons shifted the voltage dependence of the time constant of activation and decreased its voltage sensitivity, which excludes a voltage-dependent open pore block by protons as the mechanism modifying the activation curve. Changes in the extracellular pH altered the voltage dependence of steady-state inactivation and deactivation kinetics in a Ca²⁺-dependent manner, but these effects were not strictly correlated with those on activation. Model simulations suggest that protons interact with intermediate closed states in the activation pathway, decreasing the gating charge and shifting the equilibrium between these states to less negative potentials, with these effects being inhibited by extracellular Ca²⁺. Extracellular acidification also induced an open pore block and a shift in selectivity toward monovalent cations, which were both modulated by extracellular Ca²⁺ and Na⁺. Mutation of the EEDD pore locus altered the Ca²⁺-dependent proton effects on channel selectivity and permeation. We conclude that Ca²⁺ modulates T-type channel function by competing with protons for binding to surface charges, by counteracting a proton-induced modification of channel activation and by competing with protons for binding to the selectivity filter of the channel.