Related Experiment Videos
Chloride channels in myocytes from rabbit colon are regulated by a pertussis toxin-sensitive G protein
X P Sun1, S Supplisson, E Mayer
1Department of Medicine, Veteran's Affairs Wadsworth Medical Center, Los Angeles 90073.
Abstract:
Because of the high intracellular Cl- concentration ([Cl-]i) in gastrointestinal smooth muscle, receptor-mediated opening of Cl- channels at the cell resting potential could represent a plausible mechanism for initial receptor-mediated cell depolarization. To test this hypothesis, we characterized activation of large-conductance Cl- channels by the neurokinin-1 (NK-1) receptor agonist [Sar9,Met(O2)11]-substance P, by specific second messengers, and by direct G protein activation in myocytes isolated from the rabbit colon longitudinal muscle layer. In excised inside-out patches, large-conductance ion channels selective for Cl- over Na+ could be induced by holding the patch at pipette potentials values > 60 mV. The channel showed multiple smaller conductance states (< or = 20) but could open and close via a main gate. When the channel was fully open, its slope conductance was 300 pS, with substates as small as 15 pS, comparable to the predominant conductance observed in cell-attached patches. The voltage-activation profile for full conductance was bell-shaped with maximal open probability (Po) for channel opening of approximately 0 mV. In cell-attached patches, addition of the NK-1 agonist to pipette solution activated a channel that corresponded to a subconductance state of the maxi Cl- channel. The voltage-activation profile for this subconductance state showed a maximal Po value for membrane potentials of approximately 0 mV, with rapid inactivation at more positive and partial inactivation at more negative membrane potentials. In excised inside-out patches, both the full and smaller conductance states of the Cl- channel were activated by the nonhydrolyzable guanosine triphosphate analogue guanosine 5'-O-(3-thiotriphosphate) and inhibited by pertussis toxin (PTX), whereas [Ca2+]i increased channel activity only in concentrations > 1 mM. In cell-attached patches, addition of different Ca2+ ionophores resulted in channel activation in 10% of cells, and activators of protein kinase A or protein kinase C had no effect. These findings are consistent with the hypothesis of a possible role of G protein-coupled Cl- channels in receptor-mediated initial cell depolarization in longitudinal colonic smooth muscle.
Insights
Large-conductance chloride channels in colon smooth muscle may initiate cell depolarization. Neurokinin-1 receptor activation opens these channels via G protein signaling, suggesting a role in gastrointestinal muscle function.
Area of Science:
- Physiology
- Molecular Biology
- Gastroenterology
Background:
- High intracellular chloride concentration in gastrointestinal smooth muscle suggests chloride channels could mediate initial depolarization.
- Neurokinin-1 (NK-1) receptors are involved in gastrointestinal function and signaling.
Purpose of the Study:
- To investigate the role of large-conductance chloride channels in receptor-mediated depolarization of colonic smooth muscle.
- To characterize the activation mechanisms of these chloride channels by NK-1 receptor agonists and related signaling pathways.
Main Methods:
- Electrophysiological recordings (cell-attached and inside-out patch-clamp) on isolated rabbit colon myocytes.
- Characterization of channel conductance, voltage-dependence, and activation by NK-1 receptor agonist [Sar9,Met(O2)11]-substance P.
- Investigation of second messenger involvement (GTPγS, Ca2+, protein kinases) and pertussis toxin (PTX) sensitivity.
Main Results:
- A large-conductance chloride channel (300 pS) with subconductance states (down to 15 pS) was identified.
- Channel activity was voltage-dependent, with maximal open probability near 0 mV.
- NK-1 receptor agonist activated a subconductance state, suggesting G protein-coupled channel gating, sensitive to GTPγS and PTX, with minimal Ca2+ or kinase involvement.
Conclusions:
- G protein-coupled chloride channels are present in colonic smooth muscle.
- Activation of these channels by NK-1 receptor agonists contributes to initial cell depolarization.
- These findings support a role for chloride channels in receptor-mediated signaling in the gastrointestinal tract.