Regulation of the amphiuma erythrocyte NA ⁺/H ⁺ exchanger: A potential role for casein kinase-2

The Amphiuma erythrocyte Na ⁺/H ⁺ exchanger is involved in both intracellular pH and volume regulation. Although the mechanism for regulation of this exchanger remains unknown, many studies suggest a possible role of protein kinases and phosphatases in the regulation of this inducible transport system. Previous results indicated that protein kinase C and tyrosine kinases can regulate the activity of the exchanger in this red cells. More recently, the cloning and sequencing of this erythrocyte Na ⁺/H ⁺ antiport reveals several peptide sequences which are typical substrates for casein kinase-2. In view of this finding a new hypothesis has been formulated that includes the possible role of this kinase in the regulation of the exchanger. In order to test this hypothesis we have initiated the necessary studies to establish the presence of casein kinase-2 in this cells, and its role in the activation of the transport by osmotic shrinkage. Immunoblot studies indicate the presence of casein kinase-2 subunits, α and α′, in particulate and membrane fractions. No casein kinase-2 subunits were detected in the cytosol fractions consistent with the possibility of a membrane associated protein. Addition of the specific inhibitor N-(2-Aminoethyl)5-chloronaphthalene-1-sulfonamide-HCL (A-3) result in significant stimulation of the volume activated Na ⁺/H ⁺ exchanger (IC ₅₀≈50μM). Concentrations higher than 100 μM results in the reversal of this effect. Furthermore, we have also found that the addition of this casein kinase inhibitor result in a shrinkage dependent acceleration of the activation kinetics, while inactivation of the transporter is not affected by the presence of the inhibitor. These results are consistent with the possible role of casein kinase in the regulation of the Na ⁺/H ⁺ exchanger.