Under stationary conditions, opening and closing of single Torpedo electroplax chloride channels show that the number of transitions per unit time between inactivated and conducting states are unequal in opposite directions. This asymmetry, which increases with transmembrane electrochemical gradient for the chloride ion, violates the principle of microscopic reversibility and thus demonstrates that the channel-gating process is not at thermodynamic equilibrium. The results imply that the channel's conformational states are coupled to the transmembrane electrochemical gradient of the chloride ion.
Under two-electrode voltage clamp, a mutant of P. tetraurelia, restless (rst/rst), showed a large increase in induced current and an outward tail current when compared to the wild-type cell for hyperpolarizing voltage steps. An increase in the induced and tail currents is also observed for depolarizing voltage steps. The larger current during voltage steps and tail in the mutant were eliminated by the use of CsCl-filled electrodes and tetraethylammonium ion (TEA+) in the bath solution, characterizing the lesion as affecting a K+ conductance. Ionophoretic injection of ethylene glycol bis-(beta-aminoethyl ether) n,n,n',n-tetraacetic acid (EGTA) to buffer internal Ca2+ concentration reduced the increased K+ current and tail of the restless cell, indicating Ca2+ activation of the K+ current. Time course and amplitude of remaining currents after blockage of K+ conductances with Cs+ and TEA+ were similar in wild-type and restless cells suggesting no restless defect in entry of calcium. The Ca2+-activated sodium current was similar in the mutant to that in wild type arguing against a defect in calcium regulation activating the K+ channel in the restless cell. We conclude that the restless mutation alters a Ca2+-activated potassium conductance other than the one previously described. The multiplicity of Ca2+-activated potassium conductances in Paramecium is discussed.
Many G-protein-coupled receptors are only transiently active because an inactivation process stops the receptor from activating G protein molecules. Although this inactivation has been investigated in vitro, the real kinetics of the process can only be obtained from intact cells. Here we describe a method for measuring the inactivation of rhodopsin in intact photoreceptors and the application of this method to the ultraviolet rhodopsin of Limulus median eye. The results show that the inactivation process is very rapid (less than 150 ms) and occurs well before the peak of the receptor potential. We have also investigated whether the inactivation process can itself be modulated. Our results show that light-adaptation accelerates inactivation by about 10-fold, providing evidence that G-protein-mediated transduction can be modulated at this first stage.
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