G protein subunit ai-3 activates a pertussis toxin-sensitive Na+ channel from the epithelial cell line A6

HORACIO F. CANTIELLO, CHARLES R. PATENAUDE, AND DENNIS A. AUSIELLO

JOURNAL OF BIOLOGICAL CHEMISTRY

Año: 1989 p. 20867 - 20870

0021-9258

In nonpolar excitable cells, guanine nucleotide regulatory (G) proteins have been shown to modulate ion channel activity in response to hormone receptor activation. Inp olarized epithelia, hormone receptor-<; protein coupling involved in the generation of cAMP occurs on the basolateral membrane, whilep h ythseio logical response to this messenger is a stimulation of ion channel activitya t the apical membrane. In the present study we have utilized the patch-clamp technique to assess if the polarized renal epithelia, A6, have topologically distinct G proteins at their apical membrane capable of modulating Na’ channel activity. In excised inside-out patches of apical membranes, spontaneous Na+ channel activity (conductance 8-9 picosiemens) was inhibited by the addition of 0.1 mM guanosine 5’- 0-(2-thio)diphosphate to the cytosolic membrane surface without an effect on single channel conductance. In contrast, the percenot pen time of spontaneous Na+ channels increased from 6 to 50% following the addition of 0.1 mM GTP. The additiono f preactivated pertussis toxin (100 ng/ml) to the cytosolic bathing solution of the excised patch inhibited spontaneous Na+ channel activity withina minute by8 5% from approximately 47 to 7% open time and reduced the percent open time for Na+ channel activity to zero after approximately 3 min. The addition of 0.1 mM guanosine 5’-(3-O-thio)triphosphate or the addition of 20 PM purified human ai-3 subunit to pertussis toxin-treated membrane patches restored Na+ channel activity from zero to 35% open time. As little as 0.2 PM ai-3 subunit was capable of restoring Na’ channel activity. These data provide evidence for a role of pertussis toxinsensitive G proteins in the apicapl lasma membrane of renal epithelia distal to signal transduction pathways in the basolateral membranoe f these cells. This raises the possibility of a topologically distinct signal transducing pathway co-localized with the Na+ channel.cAMP occurs on the basolateral membrane, whilep h ythseio logical response to this messenger is a stimulation of ion channel activitya t the apical membrane. In the present study we have utilized the patch-clamp technique to assess if the polarized renal epithelia, A6, have topologically distinct G proteins at their apical membrane capable of modulating Na’ channel activity. In excised inside-out patches of apical membranes, spontaneous Na+ channel activity (conductance 8-9 picosiemens) was inhibited by the addition of 0.1 mM guanosine 5’- 0-(2-thio)diphosphate to the cytosolic membrane surface without an effect on single channel conductance. In contrast, the percenot pen time of spontaneous Na+ channels increased from 6 to 50% following the addition of 0.1 mM GTP. The additiono f preactivated pertussis toxin (100 ng/ml) to the cytosolic bathing solution of the excised patch inhibited spontaneous Na+ channel activity withina minute by8 5% from approximately 47 to 7% open time and reduced the percent open time for Na+ channel activity to zero after approximately 3 min. The addition of 0.1 mM guanosine 5’-(3-O-thio)triphosphate or the addition of 20 PM purified human ai-3 subunit to pertussis toxin-treated membrane patches restored Na+ channel activity from zero to 35% open time. As little as 0.2 PM ai-3 subunit was capable of restoring Na’ channel activity. These data provide evidence for a role of pertussis toxinsensitive G proteins in the apicapl lasma membrane of renal epithelia distal to signal transduction pathways in the basolateral membranoe f these cells. This raises the possibility of a topologically distinct signal transducing pathway co-localized with the Na+ channel.