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.

The gigaohm seal technique was used to study ion permeation through acetylcholine-activated channels in cell-attached patches of the extrajunctional membrane of chronically denervated, enzyme-treated cells from the sartorius muscle of the toad Bufo marinus. The most frequently occurring channel type (greater than 95% of channel openings), provisionally classified as 'extrajunctional,' had a chord conductance of approximately 25 pS under normal conditions (-70 mV, 11 degrees C, Normal Toad Ringer's). The less frequently observed channel type (less than 5% of channel openings), classified as a 'junctional' type, had a conductance of 35 pS under the same conditions, and a similar null potential. In many patches, a small percentage (usually less than 2%) of openings of the extrajunctional channel displayed a lower conductance state. The shape of the I-V curves obtained for the extrajunctional channel depended on the predominant extracellular cation. For Cs and K, the I-V curves were essentially linear over the voltage range +50 to -150 mV across the patch, suggesting that the potential independent component of the energy profile within the channel was symmetrical. For Li, the I-V curve was very nonlinear, displaying a significant sublinearity at hyperpolarized potentials. Both an electrodiffusion and a symmetrical uniform four-barrier, three-site rate-theory model provided reasonable fits to the data, whereas symmetrical two-barrier, single-site rate-theory models did not. For the alkali cations examined, the relative permeability sequence was PCs greater than PK greater than PNa greater than PLi--a "proportional" selectivity sequence. This was different from the single channel conductance sequence which was found to be gamma K greater than gamma Cs greater than gamma Na greater than gamma Li implying that ions do not move independently through the channel. The relative binding constant sequence for the channel sites was found to be a "polarizability" sequence, i.e., KLi greater than KCs greater than KNa greater than KK. There was an inverse relationship between the relative binding constant and the relative mobility for the cations examined. Under conditions when the single-channel conductance was relatively high, the conductance at depolarized potentials was lower than that predicted by both electrodiffusion and rate theory models, suggesting that there was a rate-limiting access step for ions, from the intracellular compartment into the channel.

A technical problem associated with the patch clamp technique has been the changing of solutions bathing the membrane patch. The simple technique described here solves this problem by means of a movable polythene sleeve placed on the shaft of the patch clamp pipette. The sleeve is initially placed so that the tip of the pipette is exposed. A gigaohm seal is formed using standard techniques. The patch is then excised and the sleeve is slipped down a few mm past the end of the tip of the pipette. When the pipette and sleeve is now removed from the solution, a small drop of solution covering the membrane patch is held in place at the end of the sleeve by surface tension. The pipette is then easily transferred to a different solution without passing the membrane patch through the air-water interface. The sleeve is then simply pulled back up the pipette shaft to expose the membrane patch to the new solution.

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A technique is described which permits accurate temperature control and relatively rapid temperature changes (within about 2 min for 10 degrees C changes between 10 degrees and 40 degrees C) of the solution perfusing the exposed surface of excised membrane patches. The simultaneous exchange of temperature controlled solution is also possible. Using the "sleeve technique", patches excised from cells in standard tissue culture dishes are removed to a separate chamber where temperature and solution are accurately controlled. This avoids two common limitations of existing temperature or solution control systems: (1) test solution contamination of the tissue perfusion solution which may impair cell viability and (2) the use of specialised chambers which are unsuitable for use with cultured cells. In the system described, temperature control is possible over the range of at least 4-40 degrees C. Desired temperatures can be preset to within approximately +/- 1 degrees C, and can then be controlled and measured to an accuracy of +/- 0.1 degree C. At a constant temperature, the system enables rapid solution changes, the solution bathing the excised patch being exchanged in approximately 3 s.