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Regulation of endogenous and heterologous Ca²? release-activated Ca²? currents by pH.

ABSTRACT: Deviations from physiological pH (?pH 7.2) as well as altered Ca(2+) signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca(2+) influx is the store-operated Ca(2+) entry (SOCE) or Ca(2+) release-activated Ca(2+) current (ICRAC), which is activated upon depletion of intracellular Ca(2+) stores. We here show that extracellular and intracellular changes in pH regulate both endogenous ICRAC in Jurkat T lymphocytes and RBL2H3 cells, and heterologous ICRAC in HEK293 cells expressing the molecular components STIM1/2 and Orai1/2/3 (CRACM1/2/3). We find that external acidification suppresses, and alkalization facilitates IP3-induced ICRAC. In the absence of IP3, external alkalization did not elicit endogenous ICRAC but was able to activate heterologous ICRAC in HEK293 cells expressing Orai1/2/3 and STIM1 or STIM2. Similarly, internal acidification reduced IP3-induced activation of endogenous and heterologous ICRAC, while alkalization accelerated its activation kinetics without affecting overall current amplitudes. Mutation of two aspartate residues to uncharged alanine amino acids (D110/112A) in the first extracellular loop of Orai1 significantly attenuated both the inhibition of ICRAC by external acidic pH as well as its facilitation by alkaline conditions. We conclude that intra- and extracellular pH differentially regulates ICRAC. While intracellular pH might affect aggregation and/or binding of STIM to Orai, external pH seems to modulate ICRAC through its channel pore, which in Orai1 is partially mediated by residues D110 and D112.

SUBMITTER: Beck A

PROVIDER: S-EPMC4162834 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Regulation of endogenous and heterologous Ca²⁺ release-activated Ca²⁺ currents by pH.

Beck Andreas A Fleig Andrea A Penner Reinhold R Peinelt Christine C

Cell calcium 20140802 3

Deviations from physiological pH (∼pH 7.2) as well as altered Ca(2+) signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca(2+) influx is the store-operated Ca(2+) entry (SOCE) or Ca(2+) release-activated Ca(2+) current (ICRAC), which is activated upon depletion of intracellular Ca(2+) stores. We here show that extracellular and intracellular changes in pH regulate both endogenous ICRAC in Jurkat T lymphocytes and RBL2H3 cells, and hetero ...[more]

PMID: 25168908

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Project description: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.

Dataset Information

Regulation of endogenous and heterologous Ca²? release-activated Ca²? currents by pH.

Publications

Regulation of endogenous and heterologous Ca²⁺ release-activated Ca²⁺ currents by pH.

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