Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.

Abstract

In previous papers we studied the rising phase of a miniature endplate current (MEPC) to derive diffusion and forward rate constants controlling acetylcholine (AcCho) in the intact neuromuscular junction. The present study derives similar values (but with smaller error ranges) for these constants by including experimental results from the fallins phase of the MEPC. We find diffusion to be 4 x 10-6 cm2 slightly slower than free diffusion, forward binding to be 3.3 x 107 M-s-, and the distance from an average release site to the nearest exit from the cleft to be 1.6 jtm. We also estimate the back reaction rates. Heidmann and Changeux (7).] In our approach we use the shape of the MEPC as a function of AcChoR concentration to derive forward binding rates as well as information on AcCho diffusion in the intact neuromuscular cleft. We thus complement the results obtained by chemical techniques and by single-channel recording. The derived parameters support our motivating assumptions of the "saturating disk model" used by Salpeter and colleagues (8-11) to explain the generation of the MEPC. METHODSWe recorded MEPCs by voltage clamp from the intercostal muscle of the lizard Anolis carolinensis, using experimental techniques as in the first two papers of this series (1, 2) with minor modifications.Three groups were used: (i) control muscle (esterases and receptor concentration unaltered); (ii) receptors intact and esterases fully inactivated [with 1 mM diisopropyl fluorophosphate (iPr2P-F) for 1 hr]; (iii) receptors partially inactivated [with 40 nM a-bungarotoxin (BTX) for 40 min] to -0.36 its normal value (papers 1 and 2) and esterases inactivated with iPr2P-F as in ii. The filtering conditions of the voltage clamp were modified from those used in papers 1 and 2 to improve the signal-to-noise ratio. Because the falling phase of MEPCs is slow relative to the rise time, the signals were low-pass-filtered at 1 kHz (two-pole Butterworth filter) and the digitizer sampling rate was reduced to 6.25 kHz. Even though the Nyquist frequency is only a factor of 3 above our filter cutoff, aliasing was judged to be less than 4% of peak current. All experiments were done at 22 ± 10C and -100 mV holding potential. Fall time (tf) values were obtained from the experimental MEPC as 1.23 x the 90-40% fall time. The factor 1.23 is the conversion from 90-40% to efolding time for a pure exponential. Fig. 1 shows six examples of measured MEPC traces, illustrating the three conditions used in the present study. Fig. 2 plots the relation between tf and MEPC amplitude Ac, and Table 1 gives the mean values of tf and Ac under these conditions. RESULTSWe confirm (12-15) that inactivation of esterases lengthens tf appreciably and that, when cr is reduced by BTX treatment, tf is somewhat shortened again (12, 15). Our additional observations are that, when esterases are intact, tf does not depend on Ac ( Fig. 2A), but after esterase inactivation tf increases with increasing Ac (Fig. 2B). Finally, decreasing a, with esterases inactivated, alm...

The Basement Membrane/Basal Lamina of Skeletal Muscle

“…One might imagine that the BL would block movement of acetylcholine across the synaptic cleft, but kinetic studies show that its diffusion to receptors is unimpeded (32). This result is consistent with conclusions reached from analysis of glomerular BL in kidney, which is an effective filter only for macromolecules (33).…”

Miniature endplate current rise times less than 100 microseconds from improved dual recordings can be modeled with passive acetylcholine diffusion from a synaptic vesicle.

“…4 and one corresponding to the limiting case where all ACh molecules were released instantly. D for ACh within the synaptic cleft was about 2-fold smaller than DACh, consistent with impeded movement through basal lamina material (19).…”

The difference in shape of spontaneous and uniquantal evoked synaptic potentials in frog muscle.

“…Of the many models that have been proposed to simulate synaptic currents (Magleby & Stevens, 1972a;Gage, 1976;Steinbach & Stevens, 1976;Rosenberry, 1979;Wathey, Nass & Lester, 1979;Land, Salpeter & Salpeter, 1980, 1981Land, Harris, Salpeter & Salpeter, 1984;Madsen, Edeson, Lamtt & Milne, 1984;Madsen, Edeson & Milne, 1987), those presented by Land et al (1981Land et al ( , 1984 and Wathey et al (1979) are of particular relevance to the present work, since, in these models, receptor density was one of the parameters explicitly examined. Thus, for example, Land et al (1981) concluded that, when the esterase is blocked, no correlation between miniature endplate current (MEPC) amplitude and rise time is to be expected if receptor density is high, while a positive correlation is anticipated if the density is reduced.…”

“…This prediction was borne out by their experimental results, using a-bungarotoxin to reduce the effective concentration of ACh receptors (Land et al 1981). In addition, both Land et al (1984) and Wathey et al (1979) (Sakmann, 1978;Jackson, Wong, Morris, Lecar & Christian, 1983 …”