I. Parnas

Movement of `gating charge¿ is coupled to ligand binding in a G-protein-coupled receptor

Activation by agonist binding of G-protein-coupled receptors (GPCRs) controls most signal transduction processes. Although these receptors span the cell membrane, they are not considered to be voltage sensitive. Recently it was shown that both the activity of GPCRs and their affinity towards agonists are regulated by membrane potential. However, it remains unclear whether GPCRs intrinsically respond to changes in membrane potential. Here we show that two prototypical GPCRs, the m2 and m1 muscarinic receptors (m2R and m1R), display charge-movement-associated currents analogous to ‘gating currents’ of voltage-gated channels. The gating charge–voltage relationship of m2R correlates well with the voltage dependence of the affinity of the receptor for acetylcholine. The loop that couples m2R and m1R to their G protein has a crucial function in coupling voltage sensing to agonist-binding affinity. Our data strongly indicate that GPCRs serve as sensors for both transmembrane potential and external chemical signals.

Neurotransmitter release and its facilitation in crayfish

Release and facilitated release of transmitter at neuromuscular junctions of the crayfishAstacus were measured as a function of [Ca]0 at single junctions using a patch clamp technique. Tests were made of a quantitative model that relates release of transmitter to [Ca]i. The model assumes three processes, entry of Ca during the action potential, release of transmitter as a function of [Ca]i, and removal of Ca after the action potential. Each process is decribed alternatively by linear kinetics or saturation kinetics, and predictions for different combinations of the equations are given. The main findings were in agreement with those predicted by the “saturation” model. The amplitude of synaptic current varies non-linearly with [Ca]0, log-log plot yielding a slope of about 1.6. The degree of facilitation at long intervals is an increasing function of [Ca]0. In addition, the duration of facilitation is prolonged as [Ca]0 is increased, to saturate at [Ca]0 of 9 mM.

Simultaneous Measurement of Intracellular Ca2+ and Asynchronous Transmitter Release from the same Crayfish Bouton

1 A technique has been developed to monitor neurotransmitter release simultaneously with intracellular Ca2+ concentration ([Ca2+]i) in single release boutons whose diameters range from 3 to 5 μm. 2 Using this technique, we have found a highly non‐linear relationship between the rate of asynchronous release and [Ca2+]i. The Hill coefficient lies between 3 and 4. 3 The affinity (Kd) of the putative release‐related Ca2+ receptor for asynchronous release was calculated to be in the range of 2–4 μm. 4 The same range of values of Hill coefficient and Kd were obtained when [Ca2+]i was elevated both by bath application of ionomycin and by repetitive stimulation at high frequency. 5 Our results show that the Ca2+ receptor(s) associated with asynchronous release exhibits high affinity for Ca2+.

Membrane depolarization evokes neurotransmitter release in the absence of calcium entry.

THE discovery that Ca2+ is necessary for the release of neurotransmitter, the primary means by which nerve cells communicate, led to the calcium hypothesis of neutransmitter release1–4, in which release is initiated after an action potential only by an increase in intracellular Ca2+ concentration near the release sites and is terminated (1–2 ms) by the rapid removal of Ca2+. Since then, the calcium-voltage hypothesis has been proposed5,6, in which the depolarization of the presynaptic terminals has two functions. First, in common with the calcium hypothesis, the Ca2+ conductance is increased, thereby permitting Ca2+ entry. Second, a confor-mational change is induced in a membrane molecule that renders it sensitive to Ca2+, and then binding of Ca2+ to this active form triggers release of neurotransmitter. When the membrane is repolarized, the molecule is inactivated and release is terminated, regardless of the local Ca2+ concentration at that moment. This hypothesis, in contrast to the calcium hypothesis, accounts for the insensitivity of the time course of release to experimental manipulations of intracellular Ca2+ concentation7–11, Furthermore, it explains rapid termination of release after depolarization, even though Ca2+ concentration may still be high. Here we describe experiments that distinguish between these two hypotheses and find that our results support the calcium voltage hypothesis.

Presynaptic effects of muscarine on ACh release at the frog neuromuscular junction

1 Presynaptic effects of muscarine on neurotransmitter release were studied at the frog neuromuscular junction, using focal depolarization of the presynaptic terminal to different levels. 2 Muscarine (10 μM) had a dual effect on ACh release: concomitant inhibition and enhancement of release at the same patch of presynaptic membrane. 3 These two effects were maximal at low depolarizing pulses and diminished as depolarization increased. 4 At low depolarizing pulses, atropine (1 μM) enhanced release, suggesting that ACh in the synaptic cleft causes a net tonic inhibition of ACh release. 5 In the presence of the M2 antagonist methoctramine (1 μM), muscarine (10 μM) enhanced ACh release. 6 In the presence of the M1 antagonist pirenzepine (10 μM), muscarine (10 μM) produced stronger inhibition. 7 These results show that the M2 receptor is responsible for inhibition of ACh release, while the M1 receptor is responsible for its enhancement. 8 The inhibitory effect of muscarine did not depend on extracellular [Ca2+]. Enhancement of release was abolished at low extracellular [Ca2+]. 9 The muscarine inhibitory effect was not associated with a reduction of Ca2+ current, while release enhancement was associated with an increase of Ca2+ current.

Neurotransmitter release and its facilitation in crayfish muscle

Excitatory postsynaptic currents (EPSCs) were recorded extracellularly from synaptic spots on crayfish opener muscle fibers. Synapses on the proximal fiber bundle were characterized as fast, with a relatively high quantal-release ratem of 0.2–5 and a low twin-pulse facilitationFs of 1.1–3, at 13.5 mM [Ca]0 and low (0.5/s) repetition rate. Unter the same conditions, distal “slow” synapses had a release ratem of 0.02–0.4 and a facilitationFs of 2–4. When the [Ca]0 was varied between 1.7 and 27 mM, release and facilitation were much less affected in proximal, fast synapses than in distal, slow ones. The average maximal slope of the log release to log [Ca]0 relation was 1.5 in proximal, and 3.1 in distal synapses, while the average maximal facilitationFs was 2.5 in proximal and 4.7 in distal synapses, respectively. Assuming saturation kinetics for entry of Ca into the terminal and release of transmitter, possible variations of parameters generating the fast-slow differentiation were explored. Excluding a number of possibilities, it was found that in addition to a higher maximal release level, fast synapses seem to have a higher resting [Ca]i and/or a lower cooperativity of the release mechanism, as compared to slow synapses.

The Metabotropic Glutamate G-protein-coupled Receptors mGluR3 and mGluR1a Are Voltage-sensitive

G-protein-coupled receptors play a key role in signal transduction processes. Despite G-protein-coupled receptors being transmembrane proteins, the notion that they exhibit voltage sensitivity is rather novel. Here we examine whether two metabotropic glutamate receptors, mGluR3 and mGluR1a, both involved in fundamental physiological processes, exhibit, by themselves, voltage sensitivity. Measuring mGluR3-induced K+ currents and mGluR1a-induced Ca2+-activated Cl– currents in Xenopus oocytes, we show that the apparent affinity toward glutamate decreases (mGluR3) or increases (mGluR1a) upon depolarization. Measurements of binding of [3H]glutamate to oocytes expressing either mGluR3 or mGluR1a corroborated the electrophysiological results. Using the chimeric Gα subunit, we further show that the voltage sensitivity does not reside in the G-protein. To locate sites within the receptors that are involved in the voltage sensitivity, we used chimeric mGluR1a, where the intracellular loops that couple to the G-protein were replaced by those of mGluR3. The voltage sensitivity of the chimeric mGluR1a resembled that of mGluR3 and not that of the parental mGluR1a. The cumulative results indicate that the voltage sensitivity does not reside downstream to the activation of the receptors but rather in the mGluR3 and mGluR1a themselves. Furthermore, the intracellular loops play a crucial role in relaying changes in membrane potential to changes in the affinity of the receptors toward glutamate.

Effects of intra-axonal injection of Ca2+ buffers on evoked release and on facilitation in the crayfish neuromuscular junction

Ca2+ buffers were injected into the excitatory axon of the crayfish opener muscle. The magnitude and time course of evoked release and of facilitation were measured. EGTA (on-rate about 10(6) M-1S-1) had no effect on evoked release but reduced facilitation. BAPTA and nitr-5, buffers with similar Kds but faster on-rates, reduced both evoked release and facilitation. However, these buffers had no effect on the time course of evoked release. These results show that fast Ca2+ buffers reduce the Ca2+ transient associated with evoked release and also the level of residual Ca2+ involved in facilitation. However, Ca2+ buffering is not the mechanism which controls the time course of release.

Neurotransmitter release and its facilitation in crayfish. III. Amplitude of facilitation and inhibition of entry of calcium into the terminal by magnesium.

Excitatory postsynaptic currents (EPSCs) were recorded extracellularly from synaptic spots on crayfish opener muscles. Release of transmitter was determined by counting the average number of quanta which appear after a stimulus. When [Mg]0 was increased from 2.5 to 12.5 mM, release was inhibited. Quantitatively the effect of [Mg]0 could be described by a competitive inhibition of the entry (not of the release) of Ca2+ after an impulse, with apparent dissociation constants KMg between 1.4 and 18 mM [Mg]0, assuming saturation kinetics for entry of Ca2+ and release. At constant [Ca]0, twin pulse facilitation (Fs) for short intervals (about 10 ms) increased when [Mg]0 was raised from low values, reached a maximum at a certain {ie237-1} and unexpectedly decreased again at higher [Mg]0. At higher [Ca]0, {ie237-2} shifted to higher values. This maximum of facilitation is predicted qualitatively by our theoretical model. However, the amplitude of facilitation was larger than predicted theoretically, and the {ie237-3} were smaller than predicted. The theoretical possibilities to correct these discrepancies within the framework of ‘residual calcium’ based facilitation and saturation kinetics of entry and release were analyzed, but all were in conflict with experimental findings. It is concluded that an essential element is missing in the present theory of facilitation.

Presynaptic M2 muscarinic receptors are involved in controlling the kinetics of ACh release at the frog neuromuscular junction

1 Macropatch recording was used to study release of acetylcholine in the frog neuromuscular junction evoked by either direct local depolarization or by an action potential. 2 The quantal content was established by directly counting the released quanta. The time course of release was obtained by constructing synaptic delay histograms. 3 Perfusion of the neuromuscular junction with methoctramine, a selective M2/M4 muscarinic antagonist, increased the quantal content and slowed the exponential decay of the synaptic delay histograms. Addition of the agonist muscarine reversed these effects. 4 Addition of acetylcholinesterase prolonged the decay of the delay histogram, and muscarine reversed this effect. 5 Methoctramine slowed the rise time of the postsynaptic current produced by axon stimulation without affecting either the excitatory nerve terminal current or the presynaptic Ca2+ current. 6 These results show that presynaptic M2 muscarinic receptors are involved in the process which terminates evoked ACh release.