An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes

We examined the roles of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) in calcium signaling using DT40 B lymphocytes, and a variant lacking the three IP(3)R isoforms (IP(3)R-KO). In wild-type cells, B cell receptor (BCR) stimulation activates a cation entry route that exhibits significantly greater permeability to Ba(2+) than does capacitative calcium entry. This cation entry is absent in IP(3)R-KO cells. Expression of the type-3 IP(3)R (IP(3)R-3) in the IP(3)R-KO cells rescued not only agonist-dependent release of intracellular Ca(2+), but also Ba(2+) influx following receptor stimulation. Similar results were obtained with an IP(3)R-3 mutant carrying a conservative point mutation in the selectivity filter region of the channel (D2477E); however, an IP(3)R-3 mutant in which this same aspartate was replaced by alanine (D2477A) failed to restore either BCR-induced Ca(2+) release or receptor-dependent Ba(2+) entry. These results suggest that in DT40 B lymphocytes, BCR stimulation activates a novel cation entry across the plasma membrane that depends upon, or is mediated by, fully functional IP(3)R.