Reddy M M, Kopito R R, Quinton P M
Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla CA 92093-0831, USA.
Am J Physiol. 1998 Oct;275(4):C1040-7. doi: 10.1152/ajpcell.1998.275.4.C1040.
Our objective in this study was to determine the effect of changes in luminal and cytoplasmic pH on cystic fibrosis transmembrane regulator (CFTR) Cl- conductance (GCl). We monitored CFTR GCl in the apical membranes of sweat ducts as reflected by Cl- diffusion potentials (VCl) and transepithelial conductance (GCl). We found that luminal pH (5.0-8.5) had little effect on the cAMP/ATP-activated CFTR GCl, showing that CFTR GCl is maintained over a broad range of extracellular pH in which it functions physiologically. However, we found that phosphorylation activation of CFTR GCl is sensitive to intracellular pH. That is, in the presence of cAMP and ATP [adenosine 5'-O-(3-thiotriphosphate)], CFTR could be phosphorylated at physiological pH (6.8) but not at low pH (approximately 5.5). On the other hand, basic pH prevented endogenous phosphatase(s) from dephosphorylating CFTR. After phosphorylation of CFTR with cAMP and ATP, CFTR GCl is normally deactivated within 1 min after cAMP is removed, even in the presence of 5 mM ATP. This deactivation was due to an increase in endogenous phosphatase activity relative to kinase activity, since it was reversed by the reapplication of ATP and cAMP. However, increasing cytoplasmic pH significantly delayed the deactivation of CFTR GCl in a dose-dependent manner, indicating inhibition of dephosphorylation. We conclude that CFTR GCl may be regulated via shifts in cytoplasmic pH that mediate reciprocal control of endogenous kinase and phosphatase activities. Luminal pH probably has little direct effect on these mechanisms. This regulation of CFTR may be important in shifting electrolyte transport in the duct from conductive to nonconductive modes.
本研究的目的是确定管腔和细胞质pH值变化对囊性纤维化跨膜传导调节因子（CFTR）氯离子电导（GCl）的影响。我们通过氯离子扩散电位（VCl）和跨上皮电导（GCl）监测汗腺导管顶膜中的CFTR GCl。我们发现管腔pH值（5.0 - 8.5）对cAMP/ATP激活的CFTR GCl影响很小，这表明CFTR GCl在其生理功能的广泛细胞外pH范围内得以维持。然而，我们发现CFTR GCl的磷酸化激活对细胞内pH敏感。也就是说，在存在cAMP和ATP [腺苷5'-O-(3-硫代三磷酸)]的情况下，CFTR在生理pH值（6.8）时可被磷酸化，但在低pH值（约5.5）时则不能。另一方面，碱性pH可阻止内源性磷酸酶使CFTR去磷酸化。用cAMP和ATP对CFTR进行磷酸化后，即使存在5 mM ATP，去除cAMP后CFTR GCl通常在1分钟内失活。这种失活是由于内源性磷酸酶活性相对于激酶活性增加所致，因为重新加入ATP和cAMP可使其逆转。然而，提高细胞质pH值以剂量依赖的方式显著延迟了CFTR GCl的失活，表明抑制了去磷酸化。我们得出结论，CFTR GCl可能通过细胞质pH值的变化进行调节，这种变化介导了对内源性激酶和磷酸酶活性的相互控制。管腔pH值可能对这些机制几乎没有直接影响。CFTR的这种调节在将导管中的电解质转运从传导模式转变为非传导模式方面可能很重要。
