DELINEATION OF TRANSMEMBRANE DOMAINS OF THE NA+ H+ EXCHANGER THAT CONFER SENSITIVITY TO PHARMACOLOGICAL ANTAGONISTS/

Plasma membrane Na+/H+ exchanger (NHE) isoforms NNE1 and NHE3 exhibit very different sensitivities to amiloride and its 5-amino-substituted analogues, benzoyl guanidinium derivatives (e.g. (3-methylsulfonyl-4-p iperidinobenzoyl)guanidine methanesulfonate (HOE-694)), and cimetidine . To define structural domains that confer differential sensitivity to these antagonists, unique restriction endonuclease sites were enginee red into cDNAs for each isoform near the regions that encode the putat ive membrane-spanning domains. These new sites did not modify their ph armacological properties and allowed several chimeric Na+/H+ exchanger s to be constructed by exchanging homologous segments. The modified pa rental (E1(+) and E3(+)) and chimeric molecules were stably expressed in exchanger-deficient. Chinese hamster ovary AP-1 cells and assayed f or their sensitivities to amiloride, ethylisopropylamiloride, HOE694, and cimetidine. Most chimeras showed drug sensitivities corresponding to the dominant parental segment. However, interchanging a 66-amino ac id segment containing the putative ninth transmembrane (M9) domain and its adjacent loops caused reciprocal alterations in the sensitivities of E1' and E3' to all antagonists, In addition, substituting the firs t five putative membrane-spanning domains of E3' with the correspondin g region of E1' modestly reduced the transporter's sensitivity to cime tidine but not the other compounds, These data indicate that the prote in segment between M8 and M10 mag be a major site of interaction with these antagonists, although other regions modestly influence sensitivi ty to certain drugs.