Splice Cassette II of Na+, HCO3- Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS

Activation of Na+, HCO3- cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH(i)) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na+, HCO3- cotransporter NBCn1 (slc4a7) and the Ca2+/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH(i) control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineuri A beta. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTV-VIH, the interaction was abolished. Native NBCn1 and calcineurin A beta co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na+, HCO3- cotransport activity was investigated in VSMCs of mesenteric arteries after an NH4+ prepulse. During depolarization with 50mM extracellular K+ to raise intracellular [Ca2+], Na+, HCO3- cotransport activity was inhibited 20-30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na+, HCO3- cotransport activity in VSMCs when cytosolic [Ca2+] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineuri A beta. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin A beta and the Na+/H+ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin A beta and cassette II of NBCn1. Intracellular Ca2+ activates Na+, HCO3- cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.