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J. Biol. Chem., Vol. 280, Issue 35, 30864-30872, September 2, 2005

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Localized Na⁺/H⁺ Exchanger 1 Expression Protects Ca²⁺-regulated Adenylyl Cyclases from Changes in Intracellular pH^*

Debbie Willoughby, Nanako Masada, Andrew J. Crossthwaite, Antonio Ciruela, and Dermot M. F. Cooper

From the Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom

The Ca²⁺-sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca²⁺ entry (CCE) in nonexcitable cells. The present study investigates whether this Ca²⁺-dependent modulation of AC activity is further regulated by local pH changes that can arise beneath the plasma membrane as a consequence of cellular activity. Ca²⁺ stimulation of AC8 expressed in HEK 293 cells and inhibition of endogenous AC6 in C6-2B glioma cells exhibited clear sensitivity to modest pH changes in vitro. Acid pH (pH 7.14) reduced the Ca²⁺ sensitivity of both ACs, whereas alkaline pH (pH 7.85) enhanced the responsiveness of the enzymes to Ca²⁺, compared with controls (pH 7.50). Surprisingly, in the intact cell, the response of AC8 and AC6 to CCE was largely unperturbed by similar changes in intracellular pH (pH_i), imposed using a weak acid (propionate) or weak base (trimethylamine). A range of hypotheses were tested to identify the mechanism(s) that could underlie this lack of pH effect in the intact cell. The pH sensitivity of CCE in HEK 293 cells is likely to dampen the effects of pH_i on Ca²⁺-regulated ACs and may partly explain the discrepancy between in vitro and in vivo data. However, we have found that the Na⁺/H⁺ exchanger (NHE), NHE1, is functionally active in these cells, and like AC8 (and AC6) it resides in lipid rafts or caveolae, which may create cellular microdomains where pH_i is tightly regulated. An abundance of NHE1 in these cellular subdomains may generate a privileged environment that protects the Ca²⁺-sensitive ACs and other caveolar proteins from local acid shifts.

Received for publication, December 21, 2004 , and in revised form, June 20, 2005.

^* This work was supported by the Wellcome Trust and National Institutes of Health Grant GM32483. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1PD, UK. Tel.: 44-1223-334063; Fax: 44-1223-334040; E-mail: dmfc2{at}cam.ac.uk.

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