Introduction and objectives The influx of extracellular Ca2+ into human lung mast cells (HLMC) is essential for the IgE-dependent release of preformed granule derived mediators and newly synthesised autacoids and cytokines. However the identity of the ion channels underlying the influx of Ca2+ remains unknown. Members of the recently discovered CRACM (also known as Orai) ion channel family are candidates for this function. The channels carry Ca2+ selective currents (ICRAC) that are activated when endoplasmic reticulum Ca2+ stores are emptied. Here we investigate whether CRACM channels are expressed in HLMC and their possible contribution to Ca2+ influx in these cells.
Methods HLMC were purified from lung tissue by immuno-magnetic affinity selection. Total RNA purified from cells was used in quantitative RT-PCR using TaqMan probes and primers (Applied Biosystems). For electrophysiology the whole cell method of patch clamp was used.
Results CRACM1, CRACM2 and CRACM3 mRNA transcripts were all readily detectable in HLMC. CRACM1 transcripts (4.50±0.51 copies/103 beta-actin transcripts, n=7 HLMC donors) were more abundant than CRACM2 transcripts (1.79±0.67 copies/103 beta-actin transcripts, n=5) and CRACM3 transcripts (3.33±0.95 copies/103 beta-actin transcripts, n=5). Consistent with the presence of CRACM transcripts, an ICRAC-like current was detected in HLMC following IgE-dependent activation and also in cells dialysed with 30 μM inositol triphosphate to empty endoplasmic reticulum Ca2+ stores. The Ca2+-selective current obtained under both conditions was blocked by the addition of 10 μM La3+, a known blocker of CRACM channels. Thus following addition of anti-IgE to 11 HLMC, current at −120 mV increased from −12.1±1.1 pA at baseline to −34.2±2.5 pA (p<0.0001) and following addition of 10 mM La3+ this was reduced to −17.4±2.1 pA. (p=0.0003). When HLMC were dialysed with IP3 (n=12), current increased from −15.3±1.2 pA to −40.2±3.2 pA (p<0.0001), and decreased to −17.7±1.5 pA (p<0.0001) by La3+.
Conclusions The presence of an ICRAC-like current and CRACM1, -2 and -3 mRNA transcripts in HLMC supports a role for CRACM channels in the influx of extracellular Ca2+ into these cells following activation. Such a role would make CRACM channels a potential therapeutic target in the treatment of asthma.