A study of stretch-activated channels in the membrane of frog oocytes: interactions with Ca2+ ions.
1. We have carried out patch-clamp measurements on a cationic channel in the plasma membrane of the frog oocyte, which can be specifically activated by membrane stretch. The kinetics of this channel also display a distinct dependence upon membrane potential, the probability of the channel being open increasing with membrane depolarization. 2. When the patch-clamp pipette filling solution was standard Ringer solution, the single-channel current-voltage (I-V) relationship was linear, the elementary conductance being 38 pS and the reversal potential +7 mV, suggesting very poor selectivity of the channel for the various cations. 3. The I-V relationship was highly non-linear having a strong inward-going rectification when Ca2+-free solutions were used to fill the patch pipette. These solutions also resulted in a selective, inward cationic permeability through the membrane, with K+ being more permeable than Na+ greater than Li+ greater than Ba2+ greater than Ca2+. 4. Though permeant through the stretch-activated channel, Ca2+ inhibited in a concentration-dependent manner the currents carried by other cations. La3+ (0.1 mM) was also an effective channel blocker. 5. The inward current carried by individual cations at a given membrane potential increased with increasing external cation concentration up to a saturating level, this level being maximal for K+ and minimal for Ca2+. Also the half-saturating concentration was maximal for K+ and minimal for Ca2+ at all membrane potentials. 6. In the presence of a constant Ca2+ concentration (50 microM) increasing [K+] did not change the absolute level at which the current saturated; however the half-saturating K+ concentration was greatly increased, indicating competitive inhibition between Ca2+ and K+ for the same site. 7. The data are consistent with a model based on Eyring rate theory for current conduction through ionic channels, in which we assume that the ions capable of entering the channel compete for a binding site that they must first occupy before proceeding on. The possible energy profile of the stretch-activated channel was defined by optimizing the model parameters to obtain the best fit of the experimental data. Ca2+ was found to have a smaller dissociation constant and much longer occupancy time than Na+ or K+, thus accounting for its lower permeability and inhibitory effect on current conduction by other cations through the stretch-activatable channel.
Taglietti
V
V
Institute of General Physiology, University of Pavia, Italy.
Toselli
M
M
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002412:Cations / Q000494:pharmacology; D005260:Female; D066298:In Vitro Techniques; D007473:Ion Channels / Q000502:physiology*; D008954:Models, Biological; D009865:Oocytes / Q000502:physiology*; D011312:Pressure; D011336:Probability; D011893:Rana esculenta
D002412:Cations; D007473:Ion Channels; D002118:Calcium