Ion permeation through single ACh-activated channels in denervated adult toad sartorius skeletal muscle fibres: effect of temperature

Abstract

The gigaohm seal technique was used to study the effects of temperature on ion permeation through acetylcholine-activated channels. This was done in cell-attached patches of the extrajunctional membrane of chronically-denervated, enzyme-treated cells from sartorius muscle of the toad Bufo marinus. The predominant extracellular cation in the pipette solution was Na+. Single channel current-voltage curves were measured at different temperatures and electrodiffusion and three-site-four-barrier rate theory models were used to characterize ion permeation through the channels and determine the effects of temperature on permeation parameters. The fitting of the experimental data to these models suggested the presence of at least three and probably more ion-selective sites within the channel. The most frequently occurring channel type (greater than 95% of channel openings) had a chord conductance of 25 pS at 11 degrees C and -70 mV and was classified as 'extrajunctional'. The single channel conductance of this channel had a low temperature-dependence (Q10 approximately equal to 1.3). The apparent activation enthalpy, Ea, for the conductance between 11 degrees C and 20 degrees C, did not appear to be significantly voltage-sensitive and had a value of about 17 +/- 2 kJ . mol-1 at a voltage of -70 mV. The Arrhenius plot of conductance appeared linear between 11 and 20 degrees C at all potentials examined. The data was consistent with a break in the slope of the Arrhenius plot at temperatures between 5 and 11 degrees C at all potentials examined, suggesting a possible phase transition of the membrane lipids. In contrast to the relative permeability, which was not very temperature sensitive, the relative binding constant was significantly affected by temperature. The relative Na/K binding constant sequence was: K5 degrees C greater than K20 degrees C greater than K15 degrees C much greater than K11 degrees C. In addition, the decrease in conductance observed at the most depolarized potentials was accentuated as the temperature was increased, suggesting a rate-limiting access step for ions from the intracellular solution into the channel.