Mechanism(s) underlying activation of store-operated Ca2+ entry currents, ISOC, remain incompletely understood. F-actin configuration is an important determinant of channel function, although the nature of interaction between the cytoskeleton and ISOC channels is unknown. We examined whether the spectrin membrane skeleton couples Ca2+ store depletion to Ca2+ entry. Thapsigargin activated an endothelial cell ISOC (-45 pA at -80 mV) that reversed at +40 mV, was inwardly rectifying when Ca2+ was the charge carrier, and was inhibited by La3+ (50 microM). Disruption of the spectrin-protein 4.1 interaction at residues A207-V445 of betaSpIISigma1 decreased the thapsigargin-induced global cytosolic Ca2+ response by 50% and selectively abolished the endothelial cell ISOC, without altering activation of a nonselective current through cyclic nucleotide-gated channels. In contrast, disruption of the spectrin-actin interaction at residues A47-K186 of betaSpIISigma1 did not decrease the thapsigargin-induced global cytosolic Ca2+ response or inhibit ISOC. Results indicate that the spectrin-protein 4.1 interaction selectively controls ISOC, indicating that physical coupling between calcium release and calcium entry is reliant upon the spectrin membrane skeleton.