G protein subunit, alpha i-3, activates a pertussis toxin-sensitive Na+ channel from the epithelial cell line, A6.
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
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HF
Renal Unit, Massachusetts General Hospital, Boston.
Patenaude
C R
CR
Ausiello
D A
DA
D000818:Animals; D002460:Cell Line; D004848:Epithelium; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D037342:Pertussis Toxin; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D015222:Sodium Channels; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); D037342:Pertussis Toxin; D019204:GTP-Binding Proteins