Monovalent cation permeability and Ca²⁺ block of the store-operated Ca²⁺ current I _CRAC in rat basophilic leukemia cells

Abstract.

Like voltage-operated Ca²⁺ channels, store-operated CRAC channels become permeable to monovalent cations in the absence of external divalent cations. Using the whole-cell patch-clamp technique, we have characterized the permeation and selectivity properties of store-operated channels in the rat basophilic leukemia (RBL-1) cell line. Store depletion by dialysis with InsP₃ and 10 mM EGTA resulted in the rapid development of large inward currents in Na⁺- and Li⁺-based divalent-free solutions. Cs⁺ permeated the channels poorly (P _Cs/P _Na=0.01). Trimethylamine (TMA⁺), tetramethylammonium (TeMA⁺), tetraethylammonium (TEA⁺), N-methyl-D-glucamine (NMDG⁺) and TRIS⁺ were not measurably permeant. NH₄ ⁺ was conducted well. We estimated the minimum pore diameter under divalent-free conditions to be between 0.32 nm and 0.55 nm. When cells were dialysed with buffered Ca²⁺ solution and I _CRAC activated by application of thapsigargin, P _Cs/P _Na was still low (0.08). Outward currents through CRAC channels were carried by intracellular Na⁺, K⁺ and, to a much lesser extent, by Cs⁺. Currents were unaffected by dialysis with Mg²⁺-free solution. The Na⁺ current was inhibited by external Ca²⁺ (half-maximal blocking concentration of 10 µM). This Ca²⁺-dependent block could be alleviated by hyperpolarization. The monovalent Na⁺ current was voltage dependent, increasing as the holding potential depolarized above 0 mV. Our results suggest that CRAC channels in RBL-1 cells have a smaller pore diameter than voltage-operated Ca²⁺ channels, discriminate between Group I cations, and differ markedly in their selectivity from CRAC channels reported in lymphocytes.