ABSTRACT
The trout red blood cell Na+/H+ antiporter (βNHE) displays two interesting properties: it is the only NHE known to be activated by cyclic AMP, and the activation process is followed by a desensitisation of the transport system itself.
Cloning and expression of βNHE have provided significant information about Na+/H+ activation, in particular that activation by cyclic AMP is directly dependent upon the presence of two protein kinase A consensus sites in the cytoplasmic tail of the antiporter. Expression of βNHE in fibroblasts demonstrates that the protein kinase A (PKA) and protein kinase C (PKC) activation pathways are independent and do not converge on a common kinase. Moreover, the hydrophilic C-terminal fragment is essential to the mediation of the various hormonal responses. NHE1 (the human ubiquitous isoform) is not activated by cyclic AMP, but a ‘NHE1 transmembrane domain/βNHE cytoplasmic domain’ chimera is fully activated by cyclic AMP.
In red cells, activation of βNHE is the result of phosphorylation by PKA of at least two independent sites. Desensitisation, inhibited by the phosphatase inhibitor okadaic acid, may consist of the dephosphorylation of one of these two sites. Furthermore, Calyculin A (ClA), another specific protein phosphatase inhibitor, induces in unstimulated cells a Na+/H+ exchange activity whose exchange properties are very different from those of the adrenergically stimulated antiporter. It is suggested that ClA may be able to revive ‘sequestered’ antiporters.
We propose that the molecular events underlying βNHE desensitisation could be similar to those involved in rhodopsin desensitisation. Antibodies were generated against trout red cell arrestin in order to analyse the binding of arrestin to the activated exchanger. Recombinant trout arrestin was produced in a protease-deficient strain of Escherichia coli and its functionality tested in a reconstituted rhodopsin assay.
