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State-dependent block of Orai3 TM1 and TM3 cysteine mutants: insights into 2-APB activation.
J Gen Physiol. 2014 May; 143(5):621-31.JG

Abstract

After endoplasmic reticulum (ER) Ca(2+) store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca(2+)-selective Ca(2+) release-activated Ca(2+) (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1-Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB-activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd(2+)). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB-activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd(2+)-induced block that is state dependent: Cd(2+) only blocks 2-APB-activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB-activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore.

Authors+Show Affiliations

Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

24733836

Citation

Amcheslavsky, Anna, et al. "State-dependent Block of Orai3 TM1 and TM3 Cysteine Mutants: Insights Into 2-APB Activation." The Journal of General Physiology, vol. 143, no. 5, 2014, pp. 621-31.
Amcheslavsky A, Safrina O, Cahalan MD. State-dependent block of Orai3 TM1 and TM3 cysteine mutants: insights into 2-APB activation. J Gen Physiol. 2014;143(5):621-31.
Amcheslavsky, A., Safrina, O., & Cahalan, M. D. (2014). State-dependent block of Orai3 TM1 and TM3 cysteine mutants: insights into 2-APB activation. The Journal of General Physiology, 143(5), 621-31. https://doi.org/10.1085/jgp.201411171
Amcheslavsky A, Safrina O, Cahalan MD. State-dependent Block of Orai3 TM1 and TM3 Cysteine Mutants: Insights Into 2-APB Activation. J Gen Physiol. 2014;143(5):621-31. PubMed PMID: 24733836.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - State-dependent block of Orai3 TM1 and TM3 cysteine mutants: insights into 2-APB activation. AU - Amcheslavsky,Anna, AU - Safrina,Olga, AU - Cahalan,Michael D, Y1 - 2014/04/14/ PY - 2014/4/16/entrez PY - 2014/4/16/pubmed PY - 2014/12/17/medline SP - 621 EP - 31 JF - The Journal of general physiology JO - J Gen Physiol VL - 143 IS - 5 N2 - After endoplasmic reticulum (ER) Ca(2+) store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca(2+)-selective Ca(2+) release-activated Ca(2+) (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1-Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB-activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd(2+)). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB-activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd(2+)-induced block that is state dependent: Cd(2+) only blocks 2-APB-activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB-activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore. SN - 1540-7748 UR - https://www.unboundmedicine.com/medline/citation/24733836/State_dependent_block_of_Orai3_TM1_and_TM3_cysteine_mutants:_insights_into_2_APB_activation_ L2 - https://rupress.org/jgp/article-lookup/doi/10.1085/jgp.201411171 DB - PRIME DP - Unbound Medicine ER -