title: Divergence of Ca(2+) selectivity and equilibrium Ca(2+) blockade in a Ca(2+) release-activated Ca(2+) channel.
abstract: Prevailing models postulate that high Ca(2+) selectivity of Ca(2+) release-activated Ca(2+) (CRAC) channels arises from tight Ca(2+) binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca(2+) binding for Ca(2+) selectivity in recombinant Orai3 channels, which function as highly Ca(2+)-selective channels when gated by the endoplasmic reticulum Ca(2+) sensor STIM1 or as poorly Ca(2+)-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca(2+) blocked Na(+) currents in both gating modes with a similar inhibition constant (Ki; ~25 µM). Thus, equilibrium binding as set by the Ki of Ca(2+) blockade cannot explain the differing Ca(2+) selectivity of the two gating modes. Unlike STIM1-gated channels, Ca(2+) blockade in 2-APB-gated channels depended on the extracellular Na(+) concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na(+) pore occupancy. Moreover, the second-order rate constants of Ca(2+) blockade were eightfold faster in 2-APB-gated channels than in STIM1-gated channels. A four-barrier, three-binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca(2+) and Na(+) to simulate the faster rate constants of 2-APB-gated channels qualitatively reproduces their low Ca(2+) selectivity, suggesting that ion entry and exit rates strongly affect Ca(2+) selectivity. Noise analysis indicated that the unitary Na(+) conductance of 2-APB-gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (Po; ~0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high Po state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca(2+) binding and kinetic factors contribute to high Ca(2+) selectivity in CRAC channels.
authors.affiliation: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.
authors.forename: Megumi
authors.initials: M
authors.lastname: Yamashita
authors.forename: Murali
authors.initials: M
authors.lastname: Prakriya
mesh_terms: D001665:Binding Sites; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D000071737:Stromal Interaction Molecule 1
chemical_list: D001896:Boron Compounds; D002120:Calcium Channel Agonists; D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C522867:Orai3 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D012964:Sodium; C109986:2-aminoethoxydiphenyl borate; D002118:Calcium