ORAI and store-operated calcium influx in human airway smooth muscle cells - PubMed (original) (raw)

ORAI and store-operated calcium influx in human airway smooth muscle cells

Samantha E Peel et al. Am J Respir Cell Mol Biol. 2008 Jun.

Abstract

The initial bronchoconstrictor response of the asthmatic airway depends on airway smooth muscle (ASM) contraction. Intracellular calcium is a key signaling molecule, mediating a number of responses, including proliferation, gene expression, and contraction of ASM. Ca(2+) influx through receptor-operated calcium (ROC) or store-operated calcium (SOC) channels is believed to mediate longer term signals. The mechanisms of SOC activation in ASM remain to be elucidated. Recent literature has identified the STIM and ORAI proteins as key signaling players in the activation of the SOC subtype; calcium release-activated channel current (I(CRAC)) in a number of inflammatory cell types. However, the role for these proteins in activation of SOC in smooth muscle is unclear. We have previously demonstrated a role for STIM1 in SOC channel activation in human ASM. The aim of this study was to investigate the expression and define the potential roles of the ORAI proteins in SOC-associated Ca(2+) influx in human ASM cells. Here we show that knockdown of ORAI1 by siRNA resulted in reduced thapsigargin- or cyclopiazonic acid (CPA)-induced Ca(2+) influx, without affecting Ca(2+) release from stores or basal levels. CPA-induced inward currents were also reduced in the ORAI1 knockdown cells. We propose that ORAI1 together with STIM1 are important contributors to SOC entry in ASM cells. These data extend the major tissue types in which these proteins appear to be major determinants of SOC influx, and suggest that modulation of these pathways may prove useful in the treatment of bronchoconstriction.

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Figures

<b>Figure 1.</b>

**Figure 1.

Expression of ORAI homologs in human airway smooth muscle (HASM) and siRNA-targeted knockdown of ORAI homologs. (A) mRNA expression of ORAI1, 2, and 3 in HASM cells using reverse transcriptase–polymerase chain reaction (RT-PCR) (O1: ORAI1, O2: ORAI2, O3: ORAI3). PCR products were sequenced to confirm expression. No products were detected in the RT samples (i.e., RNA samples that have not been reverse transcribed) to confirm no genomic DNA contamination. (B) Western blot analysis of ORAI1 protein expression after ORAI1 siRNA transfection; expression of smooth muscle α-actin was assessed as a control for equal protein loading. (C) siRNA-mediated knockdown of ORAI1, 2, and 3 mRNA assessed by real-time, quantitative PCR; the effects of individual siRNAs on expression of all ORAI homologs were assessed. The level of ORAI mRNA expression in HASM cells transfected with siRNA was compared relative to an untreated control, which was set to 100%. Results are expressed as the mean ± SEM, cDNA samples were tested in triplicate and the graph represents data from three separate transfections.

<b>Figure 2.</b>

**Figure 2.

Effects of ORAI1, 2, or 3 mRNA knockdown on cyclopiazonic acid (CPA)– and thapsigargin (TG)-induced Ca2+ influx. (Ai and Aii) Representative raw traces illustrating the CPA-induced changes in [Ca2+]i (presented as fluorescence intensity [FI]) in HASM cells transfected with ORAI1 siRNA (Ai) and ORAI3 siRNA (Aii), compared with HASM cells transfected with a negative control siRNA. CPA (10 μM) was added to the cells in the presence of low extracellular Ca2+ (0.1 mM) followed by the restoration of 2 mM Ca2+ as indicated. (Aiii) Summary of the data illustrated in (Ai) and (Aii) showing averaged changes in fluorescence after 2 mM Ca2+ restoration (averaged data from 8 separate experiments, at least 6 repeats within each experiment). (Bi and Bii) Representative raw traces illustrating the TG-induced changes in [Ca2+]i in HASM cells transfected with ORAI1 siRNA (Bi) and ORAI3 siRNA (Bii), compared with control cells. TG (1 μM) was added to the cells in the presence of low extracellular Ca2+ (0.1 mM) for 20 minutes before assay, followed by the restoration of 2 mM Ca2+ as indicated. (Biii) Summary of the data illustrated in (Bi) and (Bii) showing averaged changes in fluorescence after 2 mM Ca2+ restoration (averaged data from 4 separate experiments, at least 6 repeats within each experiment). Results are expressed as % changes ± SEM compared with control. Data are indicated as statistically significant with *P < 0.05 and **P < 0.01.

<b>Figure 3.</b>

**Figure 3.

Effects of ORAI1 knockdown on CPA-induced inward currents in single HASM cells assessed by whole cell patch clamp. (A) Time course of current density (inward currents measured at −80 mV, outward current measured at +80 mV); each point represents mean data ± SEM of all cells in each group; control cells treated with negative control siRNA (n = 12), ORAI1 suppressed cells (n = 17). (Bi) Representative current–voltage (I-V) relationships at point 1 from the time course. (Bii) Bar chart illustrating CPA-sensitive inward current density at point 1 (measured at −80 mV) in cells transfected with negative control or ORAI1 siRNA. Data are indicated as statistically significant with *P < 0.05.

<b>Figure 4.</b>

**Figure 4.

Inhibitory effects of 100 μM La3+, 100 μM Gd3+, and 50 μM 2-aminoethoxydiphenylborane (2-APB) on CPA-induced inward currents in single HASM cells assessed by whole cell patch clamp. (A) Time course of current density (inward currents measured at −80 mV, outward current measured at +80 mV) showing control cells versus cells treated with 100 μM La3+, each point represents mean data ± SEM of all cells in each group; control cells (n = 9), La3+-treated cells (n = 9). (Bi) Representative current–voltage (I-V) relationships at point 1 from the time course. (Bii) A bar chart illustrating CPA-sensitive inward current density at point 1 (measured at −80 mV) of control cells compared with cells treated with 100 μM La3+, 100 μM Gd3+, and 50 μM 2-APB. Data are indicated as statistically significant with *P < 0.05, **P < 0.01.

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