SUMMARY1. Properties of the Ca-activated K channel were studied in excised patches of surface membrane from cultured rat muscle cells using single channel 4. Both the frequency and effective duration of channel openings increased as the intracellular membrane surface was made more positive; the percentage of time spent in the open state increased e-fold for a 15 mV depolarization for low levels of activity.5. The percentage of time spent with 1, 2, .. .n channels open in membrane patches with n channels was described by the binomial distribution, suggesting that the channels opened and shut independently of one another.6. Single channel conductance (144 mM-K on both sides of the membrane) was essentially independent of membrane potential (-50 to + 50 mV) and [Ca]i (0 1 /zM -1 mM), but did increase with temperature, from 100 pS at 1 'C to 300 pS at 37 'C. 7. Channel activity occurred in apparent bursts, with the duration of the apparent bursts increasing with increasing [Ca]1. single channel currents. The reduced conductance state was immediately preceded and followed by a normal conducting state.10. While the kinetics of the Ca-activated K channel appear complex, its large conductance and high Ca and voltage sensitivity suggest that it is uniquely suited to resist depolarizations of the cell membrane potential that are accompanied by increases in intracellular Ca.* To whom reprint requests should be sent.

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SUMMARY1. The stochastic properties of single Ca-activated K channels in excised patches ofsurface membrane from cultured rat muscle cells were studied using the patch-clamp technique.2. 4. The distribution of all shut intervals at 0 5 /SM-Ca1 and + 30 mV was described by the sum of three exponential distributions with mean durations of: 0-21 msec (short shut distribution), 1-90 msec (intermediate shut), and 44 msec (long shut). These results indicate that the channel typically enters at least three closed channel states during normal channel activity. In addition, a few longer shut intervals not accounted for by the above distributions suggested that there was a fourth infrequently occurring inactivated closed-channel state.5

SUMMARY1. Burst kinetics of single Ca-activated K channels in excised patches of surface membrane from cultured rat muscle were studied using the patch-clamp technique.2. Channel activity was separated into bursts using a calculated gap derived from the distribution of shut intervals. Shut intervals greater than the calculated gap were taken as gaps between bursts.3. The distribution of burst duration was described as the sum of two exponential with mean durations of about 0-8 and 24 msec (1 /SM-Ca1, + 20 mV), suggesting two classes of bursts (short and long).

SUMMARY1. Desensitization of acetylcholine (ACh) receptors was studied at the frog neuromuscular junction under voltage clamp.2. ACh was applied directly to junctional receptors by stimulating the motor nerve with trains of impulses. End-plate currents (e.p.c.s) were used to estimate the total number of channel openings by the junctional ACh receptors, and miniature end-plate currents (m.e.p.c.s) were used to measure changes in post-synaptic sensitivity. Under the conditions of these experiments the changes in m.e.p.c. amplitudes were shown to be post-synaptic in origin and thus provided a measure of desensitization.3. When the acetylcholinesterase was inhibited with diisopropylfluorophosphate, neostigmine, or collagenase treatment to prolong the duration of the nerve-released ACh in the synaptic cleft, desensitization developed during repetitive stimulation of 1000 impulses at 5-33 impulses/sec and then recovered after the conditioning trains, with a time constant of about 25 sec.4. When the acetylcholinesterase was active so that the duration of ACh in the synaptic cleft resulting from each nerve impulse was brief( < 300 gsec), desensitization developed in response to 300-500 pairs of nerve stimuli if the interval between the impulses of each pair was 25 msec or less. When the interval was 30 msec or greater, however, measurable desensitization did not occur, even if the total number of channel openings was many times greater than in the experiments with shorter intervals or inhibited esterase where desensitization readily occurred.5. The desensitization observed to pairs of impulses was enhanced by chlorpromazine and decreased when the post-synaptic membrane was depolarized, properties similar to those described previously for desensitization to bath and ionophoretic application of ACh.6. These results indicate that desensitization to nerve-released transmitter is not a simple consequence of receptor activation, is not due to blockade of the open receptor channels by ACh, and does not result from ACh binding directly to desensitized receptors with a resulting shift in the receptor population towards the desensitized state.7. We suggest that the desensitization observed to nerve-released transmitter is a two-step process with both steps initiated by ACh. In the first step ACh converts some receptors into a desensitizable state which has an apparent lifetime of less than 30 msec; in the second step ACh desensitizes the desensitizable state. pmY 316 K. L. MAGLEBY AND B. S. PALLOTTA