Addition of purified human alpha i-3 subunit restored Na+ channel activity in pertussis toxin-treated apical membrane patches of A6 renal epithelia from 0% to 35% open time.
The alpha i-3 G protein subunit can directly activate pertussis toxin-sensitive Na+ channels in the apical membrane of A6 renal epithelial cells.
Absolute Event Rate: 35% vs 0%
In nonpolar excitable cells, guanine nucleotide regulatory (G) proteins have been shown to modulate ion channel activity in response to hormone receptor activation. In polarized epithelia, hormone receptor-G protein coupling involved in the generation of cAMP occurs on the basolateral membrane, while the physiological response to this messenger is a stimulation of ion channel activity at 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'-O-(2-thio)diphosphate to the cytosolic membrane surface without an effect on single channel conductance. In contrast, the percent open time of spontaneous Na+ channels increased from 6 to 50% following the addition of 0.1 mM GTP. The addition of preactivated pertussis toxin (100 ng/ml) to the cytosolic bathing solution of the excised patch inhibited spontaneous Na+ channel activity within a minute by 85% 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 alpha i-3 subunit to pertussis toxin-treated membrane patches restored Na+ channel activity from zero to 35% open time. As little as 0.2 pM alpha i-3 subunit was capable of restoring Na+ channel activity. These data provide evidence for a role of pertussis toxin-sensitive G proteins in the apical plasma membrane of renal epithelia distal to signal transduction pathways in the basolateral membrane of these cells. This raises the possibility of a topologically distinct signal transducing pathway co-localized with the Na+ channel.
Cantiello et al. (1989) studied Renal epithelia (A6 cell line). Purified human alpha i-3 subunit vs. Pertussis toxin-treated membrane patches was evaluated on Na+ channel activity (percent open time). Addition of purified human alpha i-3 subunit restored Na+ channel activity in pertussis toxin-treated apical membrane patches of A6 renal epithelia from 0% to 35% open time.