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
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
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DOI：
10.1074/jbc.m113.455386
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发表时间：
2013-03-22
影响因子：
4.8
通讯作者：
Boedtkjer, Ebbe
中科院分区：
文献类型：
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作者：
Danielsen, Andreas A.;Parker, Mark D.;Boedtkjer, Ebbe
血管平滑肌细胞（VSMCs）Na+，HCO 3-共转运的激活有助于动脉收缩过程中细胞内pH（pH（i））的控制，但涉及的信号转导途径尚不清楚。我们研究了Na+，HCO 3-协同转运蛋白NBCn 1（slc 4a 7）和Ca 2 +/钙调蛋白激活的丝氨酸/苏氨酸磷酸酶钙调神经磷酸酶之间是否存在物理和功能相互作用，并在VSMC中的pH（i）控制中发挥作用。使用酵母双杂交筛选，我们发现NBCn 1的N末端的剪接盒II与钙调神经A β相互作用。当盒II被截短或突变以破坏推定的钙调磷酸酶结合基序PTV-VIH时，相互作用被消除。天然NBCn 1和钙调磷酸酶A β从A7 r5大鼠VSMC中共免疫沉淀。肽（乙酰基-DDIPTVVIH-酰胺），它模仿假定的钙调磷酸酶结合基序，抑制免疫共沉淀，而突变的肽（乙酰基-DDIATAVAA-酰胺）没有。研究了NH_4 ~+预脉冲对肠系膜动脉平滑肌细胞Na ~+、HCO_3 ~-共转运活性的影响。在用50 mM细胞外K+去极化以提高细胞内[Ca ~（2+）]、Na ~+、HCO ~（3-）共转运活性的过程中，钙调磷酸酶抑制剂（FK_（506）和环孢素A）可抑制20-30%。当缓冲降低胞浆[Ca 2 +]时，FK 506不影响VSMC中Na+，HCO 3-共转运活性，也不破坏NBCn 1和钙神经A β之间的结合。在去甲肾上腺素诱导的动脉收缩过程中，FK 506增强了VSMCs的细胞内酸化。在VSMCs中没有观察到钙调神经磷酸酶A β和Na+/H+交换器NHE 1之间的物理或功能相互作用。总之，我们证明了钙调磷酸酶A β和NBCn 1的盒II之间的物理相互作用。细胞内Ca ~（2+）以钙调神经磷酸酶依赖的方式激活VSMCs的Na ~+、HCO ~（3-）共转运活性，这对保护VSMCs免受细胞内酸化是重要的。
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.