Differential effects of perhydrohistrionicotoxin on neurally and

Proc. Natl. Acad. Sci. USA Vol. 75, No. 3, pp. 1596-1599, March 1978

Neurobiology

Differential effects of perhydrohistrionicotoxin on neurally and iontophoretically evoked endplate currents (synaptic noise/acetylcholine receptors/ionic channel/denervated muscles/rat extensor and soleus muscles)

E. X. ALBUQUERQUE AND P. W. GAGE* Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 660 W. Redwood Street, Baltimore, Maryland 21201

Communicated by Bernhard Witkop, January 10, 1978

ABSTRACT Perhydrohistrionicotoxin, at concentrations of 10-12-10-7 M, depressed the current generated by iontophoretic application of acetylcholine to endplate regions of soleus and extensor digitorum longus muscles of rats. However, no changes in the amplitude or time course of spontaneous miniature endplate potentials or currents were seen with these concentrations of toxin. Evoked endplate currents were also unaffected by the toxin. Similarly, the responses to iontophoretic acetylcholine were depressed by these concentrations of perhydrohistrionicotoxin in chronically denervated muscles. Depression of responses in both normal and chronically denervated muscles developed gradually, was greater at higher concentrations, and was reversible. The different effects of the toxin on neurally evoked currents and currents produced by iontophoretic application of acetylcholine raise the possibility of the existence of two different populations of receptor complexes.

When acetylcholine (ACh) is secreted from a motor nerve terminal, it reacts with recognition sites in the postjunctional membrane and initiates a chain of little understood reactions leading to a conformational change in a "channel" macromolecule. The increase in cation permeability that is produced by this conformational change generates an endplate current. This sequence of events can also be initiated by externally applied ACh and Katz and Miledi (1, 2) first noticed that there were perceptible fluctuations in the endplate depolarization produced in this way. These fluctuations, which are due to variability in the number of open channels, can be analyzed to give average channel conductance and lifetime (3, 4). Because average channel lifetime, measured from power spectra of endplate current fluctuations produced by applied ACh, agrees well with the time constant of decay of neurally evoked endplate currents (4), it seems reasonable to assume that applied ACh activates the same population of channels as neurally secreted ACh. Further support for this assumption has come from experiments in which chemical agents, e.g., local anesthetics, alcohols, and atropine (5-9), had comparable effects on the time course of neurally evoked endplate currents or spontaneous miniature endplate currents (mepcs) and on the average lifetime of endplate channels measured from power spectra of ACh noise. Perhydrohistrionicotoxin (Hl2HTX) is a completely saturated analog of histrionicotoxin (10) that binds to a non-ACh receptor protein (11) termed ion conductance modulator (12). We have found that at concentrations lower than 10-6 M, H12HTX significantly depresses and eventually abolishes endplate currents elicited by iontophoretic pulses of ACh but that the toxin has little or no effect on mepcs at the same junction. The concentrations used here are lower than those tested previously (13).

This paper describes the effects of a range of H12HTX concentrations on iontophoretically evoked, spontaneous mepcs and neurally evoked endplate currents. MATERIALS AND METHODS Isolated extensor digitorum longus and soleus muscles from female Wistar rats (150-200 g) were used in these experiments. Some of these muscles were denervated in vivo 7-10 days before an experiment by techniques previously described (17). The normal bathing solution contained (mM) NaCl, 135.0; KCI, 5.0; MgCl2, 1.0; CaCl2, 2.0; NaHCO3, 15.0; Na2HPO4, 1.0; and glucose, 11.0 at pH, 7.1-7.2. Solutions flowed constantly through the tissue bath. The technique used to voltage clamp the endplate region was as described (14, 15). Current was monitored differentially across a 1 MQ resistor in series with the current electrode. The clamp potential was generally set equal to the resting membrane potential (-50 to -80 mV) in order to minimize net membrane current. ACh was ejected from a high-resistance (50-100 MQ) micropipette, filled with 2 M ACh, by passing a constant, positive current with a constant current generator (16) generally for 20-30 sec. Particular care was taken to select microelectrodes in which ACh leakage could be prevented with a small backing current (