ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation

Abstract

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca²⁺ microdomains. Here, we identified a Ca²⁺ entry component involved in Ca²⁺ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca²⁺ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca²⁺ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca²⁺ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca²⁺ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca²⁺ release through RYR1, in coordination with Ca²⁺ entry through ORAI1 and STIM, rapidly increases the number of Ca²⁺ microdomains, thereby initiating spread of Ca²⁺ signals deeper into the cytoplasm to promote full T cell activation.