Sialic acid and the surface charge associated with hyperpolarization-activated, inward rectifying channels.

The whole-cell configuration of the patch-clamp technique was used with cultured pacemaker cells from the rabbit sinoatrial node to test the hypothesis that sialic acid residues (NANA) constitute much of the negative surface charge associated with hyperpolarization-activated, inward rectifying channels. Activation-voltage relationships (between -70 and -140 mV) were determined for hyperpolarization-activated (inward rectifying) current (i(f)). Addition of 10 mM Ca2+ shifted the half-activation potential (V 1/2) from -89.5 +/- 0.9 mV to -77.9 +/- 2.6 mV (P less than 0.01), confirming the presence of negative fixed charges on the myocytes after 3 to 5 days in culture. Addition of 20 mM dimethonium, an organic divalent cation that "screens" but does not bind to negative surface charge, shifted V 1/2 from -86.8 +/- 1.4 mV to -75.0 +/- 1.7 mV (P less than 0.001) without affecting the amplitude of the current. In contrast, 10 mM Ca2+ reduced the amplitude of i(f) significantly. Incubation of cells with a highly purified preparation of neuraminidase (0.1-2.0 U/ml, 1 hr, 37 degrees C), an enzyme that selectively removes NANA from glycoproteins and glycolipids, failed to alter V 1/2 or the amplitude of i(f) significantly. Pretreatment of cells with neuraminidase (1.0 U/ml, 1 hr, 37 degrees C) failed to alter the positive shift of V 1/2 produced by dimethonium. The results suggest that NANA does not constitute the negative surface charge associated with hyperpolarization-activated, inward rectifying channels.