David M. J. Quastel

Calcium: is it required for transmitter secretion?

Ethanol multiplies miniature end-plate potential frequency independently of calcium ion concentrations and also multiplies calcium-dependent depolarization-evoked quantal release, to the same extent. This result implies a final common pathway, requiring little or no calcium, for both kinds of transmitter secretion. Chlorpromazine and hypertonicity act similarly to ethanol, but also depress depolarization-secretion coupling.

Multiple actions of zinc on transmitter release at mouse end-plates

In mouse diaphragm, the increase in frequency of mini end-plate potentials (fmepp), by Ca2+ or Ba2+ in 20 mM K+, was reversibly inhibited by Zn2+ in a manner consistent with competition between Zn2+ and Ca2+ at a site which interacts with only one atom of Zn with an apparent dissociation constant (Ki′) of about 0.015 mM. Between 0.5 mM and 2 mM, Zn2+ caused a rapid and reversible dose-dependent increasefmepp in 20 mM K+/0 Ca2+. Prolonged or repeated exposure to Zn2+ produced a slow increase infmepp followed by a decline, which once started, was not modified by of Zn2+. The time course was prolonged in raised Mg2+, bekanamycin, or in 5 mM K+ solution, and graded with Zn2+ concentration, but total numbers of MEPPs induced by 0.1 mM, 1 mM or 4 mM Zn2+ were not significantly different. Whenfmepp fell it became insensitive to Ca2+, Ba2+, La3+ (in 20 mM K+), ethanol and raised osmotic pressure. Before complete block of responses to Ca2+, the Ca2+/fmepp dose/response curve in 20 mM K+ was shifted to the right. These results indicate that Zn2+ enters the terminal via voltage-gated Ca2+ channels that interact in a complex way with these ions and then acts (a) as a partial agonist at sites where Ca2+ normally governs transmitter release, and (b) to produce irreversible changes in the nerve terminal, associated with a rise and subsequent fall offmepp and loss of sensitivity of the release mechanism to Ca2+ and other agents.

Actions of lead on transmitter release at mouse motor nerve terminals

The actions of lead (Pb2+) on transmitter release were studied at neuromuscular junctions in mouse diaphragm in vitro. The quantal content of end-plate potentials (EPPs) was reduced by Pb2+ in a dose-related manner consistent with inhibition of Ca2+ entry into nerve terminals, with a half-maximal effect at 1.4 μM (in 0.5 mM Ca2+ and 2 mM Mg2+). Pb2+ also inhibited the increased frequency of MEPPs (fMEPP where MEPPs denotes miniature EPPs) produced by Ba2+ in the presence of raised K+, blocking the calculated Ba2+ entry half-maximally at 170 μM. However, at concentrations of 50–200 nM, Pb2+ often increased fMEPP in 20 mM K+ in the presence of Ca2+ and acted to promote the irreversible effect of lanthanum (La3+) to raise fMEPP. In nominally Ca2+-free solution with 20 mM K+, brief (1 min) application of Pb2+ (20–320 μM) caused rapid dose-dependent reversible rises in fMEPP. With prolonged exposure to Pb2+,fMEPP rose and then slowly declined; after removal of Pb2+, once fMEPP had fallen to low levels, fMEPP responded nearly normally to Ca2+ or ethanol, but not to Pb2+ itself. In 5 mM K+, 0 mM Ca2+ and varied [Pb2+] (where [ ] denotes concentration), nerve stimulation caused no EPPs, but prolonged tetanic stimulation produced increases in fMEPP graded with [Pb2+] that persisted as a “tail”; results were consistent with growth fMEPP with the 4th power of intracellular Pb2+ and removal of intracellular Pb2+ with a time constant of about 30 s. These results suggest that Pb2+ acts to block the entry of Ca2+ and Ba2+ into the terminal via voltage-gated Ca2+ channels through which Pb2+, at higher concentrations, also penetrates and then acts as an agonist at intracellular sites that govern transmitter release.

Multiple potassium conductances at the mammalian motor nerve terminal

The possibility that multiple K+ conductances are present in mammalian motor nerve terminals was investigated by measuring the differential effects of tetraethyl-ammonium (TEA) and 4-aminopyridine (4-AP) on transmitter release and on nerve terminal excitability in mouse phrenic nerve-diaphram preparations. Neither 4-AP nor TEA alter the spontaneous frequency of miniature end-plate potentials (fmepp) and therefore evidently do not affect calcium channels directly. Both 4-AP and TEA increased the quantal content of end-plate potentials (epps) evoked by nerve stimulation but the effects were not mutually exclusive; TEA continued to act in the presence of a maximally effective concentration of 4-AP. The increase in transmitter release evoked by focal polarisation of the terminal was not effected by 4-AP, whereas TEA exerted an effect consistent with a reduction in membrane conductance. Similary, threshold for nerve terminal action potential generation was not affected by 4-AP, but TEA caused a reduction in threshold and alteration of the ‘accommodation curve’ indicative of a reduction of membrane conductance. Under conditions where one would predict no contribution of calcium K+ conductance, i.e., when release was evoked by Ba2+ in the absence of Ca2+, the different and non-competing effects of TEA and 4-AP were still apparent. It is concluded, therefore, that the motor nerve terminal possesses at least two K+ conductances, not including calcium activatedgK, which may be distinguished pharmacologically.

Anion permeability of motor nerve terminals

Motor nerve terminals in mouse and frog display behavior consistent with an appreciable permeability of the nerve terminal membrane to chloride. In mouse diaphragm, in the presence of 15 mM K+ and 2 mM or 8 mM Ca2+, replacement of Cl− by NO3−, Br− or acetate causes a transient increase in the quantal release of acetylcholine, measured as the frequency of spontaneously occurring miniature end plate potentials (FMEPP); a rapid rise in FMEPP is followed by a slow decline, with a half-time of about 4 min, to an equilibration level close to the control level. After equilibration in a solution in which the Cl− is replaced by another anion, return to Cl−-containing solution causes a transient decrease in FMEPP with a subsequent slow recovery. The data are consistent with transient nerve terminal depolarization or hyperpolarization, reflecting a nerve terminal permeability to anions in the sequence Cl−>Br−>NO3−>acetate. In 5 mM K+, changes in nerve terminal excitability, determined using focal stimulation, are also consistent with alteration of nerve terminal membrane potential as a consequence of anion substitution. The time course of relaxation of FMEPP after a change from Cl− to an anion of lower permeability, or vice versa, is considerably slower than that expected if Cl− permeability of nerve terminals is similar to that of skeletal muscle fibres, and if the nerve terminal behaves as a single compartment. In frog cutaneous pectoris, transient changes in FMEPP produced by substitution of anions in the bathing solution were similar to those produced in mouse diaphragm, but more rapid in time course.