CONDUCTANCE AND KINETIC-PROPERTIES OF SINGLE NICOTINIC ACETYLCHOLINE-RECEPTOR CHANNELS IN RAT SYMPATHETIC NEURONS

1. The unitary conductance of nicotinic acetylcholine (ACh) receptor channels in rat sympathetic neurones has been studied. Conductance estimates varied from 26-48 pS with a mean of 36.8 pS in 1 mM-Ca2+. The main conductance level varied from patch to patch and the presence (or absence) of additional conductance levels also varied. 2. The channels showed large open channel noise and experiments with 300 mM-NaCl in the patch pipette substantially increased the open channel noise. The appearance of detectable step-like transitions within this noise strongly suggested the existence of closely spaced discrete levels. 3. Removal of divalent cations from the external solution increased the unitary channel conductance. Altering the main permeant ion in divalent-free solutions gave the following conductance sequence: K+ (93 pS) > Cs+ (61 pS) > Na+ (51 pS) > Li (23 pS). 4. Replacement of Na+ by Cs+ in the external solution considerably reduced the current evoked by ACh in whole-cell recordings and the channel-opening frequency in outside-out patches. 5. The kinetic properties of channels activated by ACh and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) were also studied. At low concentrations of ACh and DMPP the gap distributions were complex and best fitted by the sum of four exponential components. Individual activations (bursts) were interrupted by the two shortest closed periods the briefer of which had time constants of 36-mu-s for ACh and 67-mu-s for DMPP. 6. The distribution of burst lengths had two components for each agonist, each component making up about 50% of the total area under the distribution. For ACh, the time constant of the longer component (12.2 ms) was similar to the decay time constant of excitatory postsynaptic potentials (EPSCs) at similar temperature and potential. For DMPP the time constant of the longer component was 17.6 ms. 7. The relative number of brief gaps per long burst was much larger for ACh than for DMPP. Therefore the corrected mean open time for ACh (0.86 ms) was much shorter than that for DMPP (2.3 ms). 8. In terms of receptor mechanism, the values of the channel opening equilibrium constant (beta/alpha) estimated from these numbers (ACh, 23; DMPP, 25) suggest that both agonists are efficaceous. 9. DMPP is a potent blocker of the channel with an equilibium dissociation constant (K(B) of around 50-mu-M and blockage gaps of around 1 ms duration. ACh also blocks the channel but with a higher K(B) of around 470-mu-M. 10. The equilibrium concentration-response relationship was estimated for a range of ACh concentrations by measuring the proportion of time a channel stays open (p(o)) during the clusters of opening that occur on escape from desensitization. The p(o) values for clusters at a given ACh concentration were found to be very variable, however, division of clusters into 'low p(o)' and 'normal' allowed a plausible activation mechanism to be fitted to the data. 11. A randomization test suggested that the existence of such heterogeneity is due to more than one population of clusters made up of channels with quite different open and closed times. It is not known, however, whether such differences are due to the existence of more than one channel type or to variation in the behaviour of a single channel type with time.