Extracellular Ca2+ Modulates the Effects of Protons on Gating and Conduction Properties of the T-type Ca2+ Channel α1G (CaV3.1)

Abstract

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 α1G (CaV3.1) 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.

Two sets of amino acids of the domain I of Cav2.3 Ca2+ channels contribute to their high sensitivity to extracellular protons

“…In the Cav1.2 subunit, these glutamates can be protonated at acidic pHo leading to partial block of ion fluxes and thus to inhibition of the macroscopic current [9,20]. A protondependent decrease of channel selectivity has also been described in LVA T-type Ca 2+ channels, which have, however, a different selectivity filter [11,28]. Here, we demonstrate that pHo-induced variations of current amplitude can be in part attributed to changes in the channel conductance; however, the fact that extracellular protons affect differentially the conductance of Cav2.1 and 2.3 (Figs.…”

Evidence for common structural determinants of activation and inactivation in T-type Ca2+ channels

“…This particular model was first developed by Burgess et al [1] and used later to describe the basal properties of Ca V 3.1 as well as the effects of extracellular pH and Ca 2+ [35], mutations in the selectivity filter [36], and arachidonic acid [37]. Similar models have been used for native T-type channels [7,9] and all Ca V 3 isoforms expressed in heterologous systems [1,11,31].…”

“…The gating properties of T-type channels have been described by several models that share the same general features, including partial coupling between macroscopic inactivation and activation [1,3,7,9,11,16,31,[35][36][37]. Among these models, we decided to use the one shown in Fig.…”

“…Among these models, we decided to use the one shown in Fig. 1a, as it has been refined to describe the basal properties of Ca V 3.1 in our experimental conditions, as well as the gating modifications induced by mutations [36] and several channel modulators [35,37].…”

“…Parameters b and c, were determined from the fit of the voltage dependence of tail current amplitudes (see Supplementary information, Fig. S1; [35,36]). Average I-V relationship of Ca V 3.1 (N=11) was best fit with V act = −43.0±0.6 mV and s act =5.14±0.14 mV, whereas for M1532I (N=7) and GΔC 1 (N=7) V act =−50.2±1.6 mV and s act =7.2± 0.4 mV and V act =−44.9±1.3 mV and s act =5.95±0.13 mV, respectively (Fig.…”