Reddy M M, Quinton P M
Division of Biomedical Sciences, University of California, Riverside 92521, USA.
Am J Physiol. 1996 Jul;271(1 Pt 1):C35-42. doi: 10.1152/ajpcell.1996.271.1.C35.
Previously, we showed in the native sweat duct that, in the presence of 0.1-0.5 mM ATP, nonhydrolyzable ATP analogue adenosine 5'-adenylylimidodiphosphate (AMP-PNP) can activate cystic fibrosis transmembrane conductance regulator Cl- conductance (CFTR GCl) (15). The objective of this study is to determine if 1) nonhydrolytic ATP binding alone can activate CFTR GCl after stable phosphorylation [in the presence of adenosine 5'-O-(3-thiotriphosphate) and phosphatase inhibition cocktail] of CFTR or 2) an ATP hydrolysis (in addition to phosphorylation) is required to support subsequent nonhydrolytic ATP regulation of CFTR GCl. We show that stably phosphorylated CFTR could only be activated by AMP-PNP in the presence of a small background ATP concentration. However, AMP-PNP can sustain previously activated CFTR GCl in the absence of ATP, even though Mg2+ is required for phosphorylation activation of CFTR GCl. However, once stably phosphorylated, ATP activation of CFTR GCl is independent of Mg2+. Our results show that both hydrolytic and nonhydrolytic interactions regulate CFTR GCl in vivo. Nonhydrolytic ATP interaction plays a significant role in both activation and deactivation of CFTR GCl.
此前，我们在天然汗腺导管中发现，在存在0.1 - 0.5 mM ATP的情况下，不可水解的ATP类似物腺苷5'-腺苷酰亚胺二磷酸（AMP-PNP）可激活囊性纤维化跨膜传导调节因子Cl-电导（CFTR GCl）（15）。本研究的目的是确定：1）在CFTR稳定磷酸化后（在存在腺苷5'-O-(3-硫代三磷酸)和磷酸酶抑制混合物的情况下），仅非水解性ATP结合能否激活CFTR GCl；或2）是否需要ATP水解（除磷酸化外）来支持随后对CFTR GCl的非水解性ATP调节。我们发现，在存在少量背景ATP浓度的情况下，稳定磷酸化的CFTR仅能被AMP-PNP激活。然而，即使Mg2+是CFTR GCl磷酸化激活所必需的，AMP-PNP在不存在ATP的情况下仍能维持先前激活的CFTR GCl。然而，一旦稳定磷酸化，CFTR GCl的ATP激活就与Mg2+无关。我们的结果表明，水解和非水解相互作用在体内均调节CFTR GCl。非水解性ATP相互作用在CFTR GCl的激活和失活中均起重要作用。
