Experiments were performed to test the applicability of permeability kinetics to whole frog sartorius muscle using K ¢ ions as tracers of potassium flux. The whole muscle was found to obey closely the kinetic laws expected to hold for single cellular units in which the potassium fluxes are membrane-limited and intracellular mixing is rapid enough not to introduce serious error. In a 5 mM K Ringer's solution, potassium effiux was very nearly equal to influx when the rate constant for K a loss was applied to the whole of the muscle potassium. Over a fairly wide range of external potassium concentration, the assumed unidirectional fluxes measured with tracer K a showed good agreement with net potassium changes determined analytically. The specific activity of potassium lost from labeled muscles to an initially K-free Ringer's solution was measured as a test of the adequacy of intracellular mixing. The results were those expected for a population of cells with uniformly distributed intracellular K ¢. A small deviation was encountered which can be attributed either to a dispersion of fiber sizes in the sartorius or to a possible small additional cellular compartment in each individual fiber. The additional cellular compartment, should it exist, contains from 0.5 to 1 per cent of the muscle potassium. This is evidently not large enough to interfere seriously with the applicability of permeability kinetics to the whole muscle. I N T R O D U C T I O NI o n fluxes have been previously measured in whole muscle b y m a n y investigators. T h e work has led to two main kinetic models for whole muscles. O n e model regards whole muscle as a population of single cellular units each obeying simple first-order permeability kinetics (Keynes, 1954; Creese, 1954). According to this model, the whole muscle will, with possible minor p e r t u r b ations, o b e y the same kinetics. T h e other model proposed regards whole muscle as an assemblage of cellular units in which simple permeability kinetics are not obeyed due to the presence of large perturbations caused b y slow diffusion intracellularly (Harris, 1957; Harris and Steinbach, 1956). For con-605

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The potassium exchange properties of glycerol-treated sartorius muscles of the frog were determined. Potassium (K) uptake, efflux, and net flux were measured in the presence of glycerol and at various times after exposure to glycerol and return to isotonic Ringer solution. Potassium uptake was not altered by the presence of glycerol but was reduced on the average 53 % after glycerol treatment. Efflux transiently increased in the presence of glycerol and was reduced 37 % after glycerol removal. Consequently, there was a net loss of intracellular potassium as well as a gain of sodium. In contrast to the irreversible alterations of potassium exchange induced by glycerol treatment, action potentials with normal negative afterpotentials (N.A.P.) were elicited 4-5 hr after glycerol removal. The reappearance of the N.A.P. was associated with a return of the membrane potential to normal values (90 4 2 my). However, the response of these muscles to reduced extracellular potassium was anomalous. In K+-free Ringer solution the average resting membrane potential was 74 4-3 my and a positive afterpotential of 11 + 3 mv was associated with the action potential.

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The effects of lobeline and tubocurarine on the voltage-clamped endplates of frog sartorius and cutaneous pectoris muscles were examined at room temperature (20-230C The ability of lobeline to shorten ti/2 and to remove the voltage dependence of ti/2 was partially antagonized by Mg2+ (13 mM) As expected, when lobeline or tubocurarine was removed from the bath or when acetylcholine release from the motor nerve terminals was increased by 4-aminopyridine (20 AM) and Ca2+ (10 mM) (in the presence of lobeline or tubocurarine), the amplitude of e.p.c.s increased as a function of time. However, the ti/2 of the decay phase of the e.p.c.s remained shortened (i.e., unaltered from the earlier treatment). These results suggest that both tubocurarine and lobeline have at least two distinct postjunctional actions including: (j) a block of the acetylcholine receptor and (ii) a block of the ionic channel associated with the acetylcholine receptor. Tubocurarine acts both on the acetylcholine (AcCho) receptor and on the AcCho receptor-activated ionic channel at the neuromuscular junction (1-3) to block transmission. The neuromuscular blocking properties of lobeline (4,5) suggest that lobeline, like tubocurarine, may have blocking actions both at the receptor and at the receptor-activated ionic channels. We compared the actions on endplate currents (e.p.c.s) of lobeline with those of tubocurarine and found that both lobeline and tubocurarine act on the ionic channel.In addition, we were able to discriminate between the actions of the drugs on the AcCho receptor and on the ionic channels. By increasing AcCho release, we could selectively antagonize the blocking action of lobeline and tubocurarine on the receptor without affecting the action on the ionic channel. Conversely, in the case of lobeline, we found that Mg2+ selectively antagonizes the blockade' of the ionic channel. Furthermore, by studying the washout of lobeline and tubocurarine from the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5003endplates, we demonstrated that the reversal of blockade of the receptor occurs long before the reversal of the blockade of the channel. Some results have been reported (6, 7). METHODS Drugs used were lobeline-HCI (Sigma), tubocurarine (Sigma), and 4-aminopyridine-(4-AP) (J. T. Baker).All the experiments reported here were done on voltageclamped endplates of frog (Rana pipiens) sartorius or cutaneous pectoris muscles at 20-230C in Ringer s solution of the following composition (mM): NaCl, 98.5; KCI, 2.5; CaCl2, 2; NaH2PO4, 1.9; Na2HPO4, 4.8. Ringer's solution containing 10 mM Ca2+ had the following composition (mM): NaCl, 98.5; KCI, 2.5; CaC12, 10; Tris, 2.0.The two-microelectrode voltage clamp described by Deguchi and Narahashi (8) Where mean values of several experiments were presented, the standard error of the mean is included. RESULTSLobeline (50 AM) and tu...

The effect of two commonly used sodium substitutes, tris and glucosamine, on the amplitude and kinetics of miniature end-plate currents (MEPCs), acetylcholine (ACh) induced end-plate currents (EPC) and EPC fluctuations was studied in voltage clamped single muscle fibres from a monolayer preparation of the cutaneous pectoris muscle. Total replacement of sodium with each substitute shifted the reversal potential from -4.7 mV (normal sodium solution) to -3.6 mV (tris) and -49.0 mV (glucosamine). In tris and glucosamine substituted solutions the current (MEPC or EPC) - voltage relation became markedly nonlinear, with peak current decreasing with membrane hyperpolarization. Peak current at +40 mV, was unaltered in tris solutions and reduced in glucosamine substituted solutions. MEPCs decayed with a single exponential time course and the EPC fluctuation spectra were characterized by single Lorentzian functions in both normal sodium solution and each substituted solution. Analysis of EPC fluctuations demonstrated that both tris and glucosamine decrease single channel conductance and increase channel lifetime. Both effects were enhanced by either membrane hyperpolarization or by increasing the concentration of each substitute. In the presence of each cationic substitute, single channel conductance increased and mean channel lifetime decreased with membrane depolarization. Analysis of the data according to the constant field assumptions (Goldman, Hodgkin, Katz equation) provided an inadequate description of experimental currents obtained at hyperpolarized membrane potentials with total ion substitution. Shifts in reversal potential with partial substitution were, however, adequately predicted by the GHK equation. These results suggest that tris and glucosamine ions interact with end-plate channels to reduce cation permeability and decrease channel closing rates. The dependence of this block on the level of membrane potential suggests that these cations bind to site(s) within open end-plate channels.

Nicardipine and other calcium channel effectors (CCEs) were studied for their effects on nicotinic acetylcholine receptor (nAChR) activity. While CCEs had no effect on frog (Rana pipiens) skeletal muscle contractions resulting from nerve stimulation or direct stimulation of the muscle, nicotinic agonist-induced contractures were blocked. Nicardipine did not affect nAChR single-channel open time or amplitude, corroborating data from endplate currents (EPCs); EPC amplitudes and decays were unaffected. All the CCEs tested, however, non-competitively blocked nAChRs. The block of nAChRs resulted in a shortened agonist-induced depolarization and thus a diminished contracture response. An increase in cultured mouse skeletal muscle (C-2) cell single-channel closed times was observed with the intracellular addition of nicardipine, verifying a direct block of nAChRs. Using single-channel analysis, nicardipine's site of action, or at least access to its site of action, was determined to be at the intracellular side of the receptor. A direct action of the CCEs on the nAChR was also shown by their ability to block phencyclidine (PCP) binding to Torpedo nobiliana membranes. All the CCEs blocked specific binding of [3H]-PCP to its binding site on the nAChR-channel complex, with bepridil and nicardipine being the most potent. These data are compatible with a model in which nicardipine and other CCEs, at concentrations which do not alter nAChR channel open time or conductance, block the effects of superfused nicotinic agonist on nAChRs either by stabilizing the formation of the nAChR desensitized state or by effecting a slow channel block.