The polybasic lysine-rich domain of plasma membrane-resident STIM1 is essential for the modulation of store-operated divalent cation entry by extracellular calcium

STIM1 acts as an endoplasmic reticulum Ca2 + sensor that communicates the filling state of the intracellular stores to the store-operated channels. In addition, STIM1 is expressed in the plasma membrane, with the Ca2 + binding EF-hand motif facing the extracellular medium; however, its role sensing extracellular Ca2 + concentrations in store-operated Ca2 + entry (SOCE), as well as the underlying mechanism remains unclear. Here we report that divalent cation entry stimulated by thapsigargin (TG) is attenuated by extracellular Ca2 + in a concentration-dependent manner. Expression of the Ca2 +-binding defective STIM1(D76A) mutant did not alter the surface expression of STIM1 but abolishes the regulation of divalent cation entry by extracellular Ca2 +. Orai1 and TRPC1 have been shown to play a major role in SOCE. Expression of the STIM1(D76A) mutant did not alter Orai1 phosphoserine content. TRPC1 silencing significantly attenuated TG-induced Mn2 + entry. Expression of the STIM1(K684,685E) mutant impaired the association of plasma membrane STIM1 with TRPC1, as well as the regulation of TG-induced divalent cation entry by extracellular Ca2 +, which suggests that TRPC1 might be involved in the regulation of divalent cation entry by extracellular Ca2 + mediated by plasma membrane-resident STIM1. Expression of the STIM1(D76A) or STIM1(K684,685E) mutants reduced store-operated divalent cation entry and resulted in loss of dependence on the extracellular Ca2 + concentration, providing evidence for a functional role of plasma membrane-resident STIM1 in the regulation of store-operated divalent cation entry, which at least involves the EF-hand motif and the C-terminal polybasic lysine-rich domain.

► Store-operated divalent cation entry is modulated by extracellular Ca2+.
► STIM1(D76A) abolishes the regulation of cation entry by extracellular Ca2+.
► The STIM1(K684,685E) mutant impairs the STIM1-TRPC1 association.
► The STIM1(K684,685E) mutant abolishes the regulation of cation entry by Ca2+.
► The polybasic domain of STIM1 is essential for the regulation of Ca2+ entry.