THE CYTOPLASMIC DOMAIN OF THE NA+ H+ EXCHANGERS (NHES) DICTATES THE NATURE OF THE HORMONAL RESPONSE - BEHAVIOR OF A CHIMERIC HUMAN NHE1/TROUT BETA-NHE ANTIPORTER/

Studies of the effect of cAMP on the cloned Na+/H+ exchangers (NHEs) a re difficult to interpret as variable results have been reported for t he different isoforms when expressed in various cell types. We took ad vantage of the fact that the human NHE1 and the trout erythrocyte beta NHE, when expressed in the same cell line, PS120, respond differently to cAMP (NHE1 is insensitive, beta NHE is activated) to analyze the m olecular mechanisms of cAMP activation. We constructed both a chimera between NHE1 and beta NHE and a set of beta NHE mutants to delineate t he critical parts of the molecule involved in the activation process. NHE1 becomes cAMP stimulated when its cytoplasmic domain is replaced b y the cytoplasmic domain of beta NHE; thus, the cytoplasmic C terminus of beta NHE, which contains two cAMP-dependent consensus sequences, i s essential to confer cAMP dependence. Serine to glycine substitution of only one of the two protein kinase A (PKA) consensus sites decrease d by 60% the ability of cAMP to activate Na+/H+ exchange. Simultaneous Ser to Gly substitution of the two PKA consensus sites decreased the cAMP mediated activation by 72%. The residual activation required a cy toplasmic fragment (aa 559-661) that contains four sequences considere d likely as putative PKA consensus sites. The results obtained with th e chimeric NHE also demonstrated that if the cytoplasmic C terminus is crucially involved in the hormonal activation, the rate of Na+/H+ exc hange so induced can be modulated by the nature of the interaction bet ween the N- and C-terminal domains.