Biophysical and Pharmacological Characterisation of Calcium Channels in Human Mast Cells

Abstract

The best characterised calcium channel in immune cells is the store-operated calcium release activated calcium channel (CRAC); studies in heterologous expression systems and knockout mice have identified Orai and its regulator STIM1, an ER-resident calcium sensor, as the molecular constituents of CRAC. Members of the TRPC family are also likely to contribute to store-operated calcium entry (SOCE) in immune cells. The expression of Orai and TRPC channels in primary human mast cells has not yet been investigated, but is of key importance given the critical role these cells play in the pathophysiology of type-1 hypersensitivity reactions. Allergic activation occurs following cross-linking of the FcεRI receptor by antigen-IgE complexes that, via PLC-γ signalling, leads to robust activation of SOCE. The resulting calcium signal stimulates secretion and production of pro-inflammatory mediators. The aim of this study was to characterize the biophysical and pharmacological properties of CRAC channels in human lung mast cells (HLMC) following SOCE. RT-PCR revealed mRNA expression for Orai1, Orai2, STIM1 and STIM2 but not Orai3 in HLMCs. Whole-cell patch clamp recordings of isolated, cultured HLMCs were carried out using a Cs-based internal solution, with added IP3 and BAPTA to activate SOCE. These conditions activated an inwardly rectifying current with Erev= 18±4mV (n=6), a value less positive than expected for a pure Orai/CRAC current. However, the current was potentiated in divalent-free solution 563.7±6% (n=6), as expected for Orai/CRAC currents, and was significantly attenuated 51.4±8% (n=6), by the Orai-specific inhibitor Synta66 (10μM) (Ng et al. 2008., Di Sabatino et al. 2009). Calcium imaging in HLMCs loaded with fura-2 AM showed that Synta66 also attenuated anti-IgE induced calcium signaling by 62.2%(n=14). Taken together the data suggest that both Orai and TRPC channels contribute to SOCE in HLMCs.