Divergence of Ca2+ selectivity and equilibrium Ca2+ blockade in a Ca2+ release-activated Ca2+channel

Megumi Yamashita, Murali Prakriya*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

28 Scopus citations


Prevailing models postulate that high Ca2+ selectivity of Ca2+ release-activated Ca2+ (CRAC) channels arises from tight Ca2+ binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca2+ binding for Ca2+ selectivity in recombinant Orai3 channels, which function as highly Ca2+-selective channels when gated by the endoplasmic reticulum Ca2+ sensor STIM1 or as poorly Ca2+-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca2+ blocked Na+ currents in both gating modes with a similar inhibition constant (Ki~25 μM). Thus, equilibrium binding as set by the Ki of Ca2+ blockade cannot explain the differing Ca2+ selectivity of the two gating modes. Unlike STIM1-gated channels, Ca2+ 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 Ca2+ 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 Ca2+ and Na+ to simulate the faster rate constants of 2-APB-gated channels qualitatively reproduces their low Ca2+ selectivity, suggesting that ion entry and exit rates strongly affect Ca2+ 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 Ca2+ binding and kinetic factors contribute to high Ca2+ selectivity in CRAC channels.

Original languageEnglish (US)
Pages (from-to)325-343
Number of pages19
JournalJournal of General Physiology
Issue number3
StatePublished - Mar 2014

ASJC Scopus subject areas

  • Physiology

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