Protein carboxyl methylation controls intracellular pH in human platelets

    loading  Checking for direct PDF access through Ovid


ObjectivesCarboxyl methylation is a reversible post-translational event which regulates the function of several cellular proteins. Because the human Na+–H+ antiporter (NHE-1) possesses a C-terminal consensus sequence for carboxyl methylation, we examined the role of protein carboxyl methylation in the regulation of intracellular pH homeostasis.DesignExperiments were conducted using human platelets and N-acetyl-S-trans, trans-farnesyl-L cysteine (AFC), a specific prenylcysteine methyltransferase inhibitor. The effect of AFC on both basal intracellular pH (pHi) and on the kinetic properties of the Na+–H+ antiporter was characterized.Materials and methodspHi was determined in cell suspensions using 2,7-biscarboxyethyl-5(6)-carboxy-fluorescein tetraacetoxymethyl ester, a fluorescent pH indicator. The kinetics properties of the Na+–H+ antiporter activity were determined using platelets acidified with nigericin and challenged with varying extracellular concentrations of Na+.ResultsAFC (20 μmol/l) decreased basal pHi significantly (7.047 ± 0.011 versus 7.133 ± 0.012 for control, P < 0.001). The acidification was dose-dependent and reached steady state 3 min after AFC addition. In the absence of extracellular Na+, the platelets were acidified to the same extent with AFC or with ethanol (control): 6.530 ± 0.031 versus 6.532 ± 0.031 (P = 0.97). However, upon addition of Na+, the platelets treated with AFC showed a significant decrease in the maximal value for initial pHi recovery compared with controls: 0.788 ± 0.041 versus 0.983 ± 0.047 pH/min (P < 0.02). AFC also increased the Hill coefficient (2.89 ± 0.22 versus 2.14 ± 0.16, P < 0.03), and tended to decrease K0.5, the [Na+] corresponding to half-maximal activation (51.3 ± 1.8 versus 60.5 ± 3.9 μmol/l, P = 0.06) of the antiporter.ConclusionOur data indicate that inhibition of carboxyl methylation reduces basal pHi and alters the kinetic properties of the Na+–H+ antiporter in human platelets, suggesting that carboxyl methylation is implicated in the regulation of intracellular pH homeostasis.

    loading  Loading Related Articles