α-Conotoxin M1 (CTx) blocks αδ binding sites of adult nicotinic receptors while ACh binding at αε sites elicits only small and short quantal synaptic currents.

In 'embryonic' nicotinic receptors, low CTx concentrations are known to block only the αδ binding site, whereas binding of ACh at the αγ-site elicits short single channel openings and short bursts. In adult muscles the αγ- is replaced by the αε-site. Quantal EPSCs (qEPSCs) were elicited in adult muscles by depolarization pulses and recorded through a perfused macropatch electrode. One to 200 nmol L(-1) CTx reduced amplitudes and decay time constants of qEPSCs, but increased their rise times. CTx block at the αδ binding sites was incomplete: The qEPSCs still contained long bursts from not yet blocked receptors, whereas their average decay time constants were reduced by a short burst component generated by ACh binding to the αε-site. Two nanomolar CTx applied for 3 h reduced the amplitudes of qEPSCs to less than half with a constant slope. The equilibrium concentration of the block is below 1 nmol L(-1) and lower than that of embryonic receptors. CTx-block increased in proportion to CTx concentrations (average rate 2 ×: 10(4) s(-1)·mol(-1) L). Thus, the reactions of 'embryonic' and of adult nicotinic receptors to block by CTx are qualitatively the same. - The study of the effects of higher CTx concentrations or of longer periods of application of CTx was limited by presynaptic effects of CTx. Even low CTx concentrations severely reduced the release of quanta by activating presynaptic M2 receptors at a maximal rate of 6 ×: 10(5) s(-1)·mol(-1) L. When this dominant inhibition was prevented by blocking the M2 receptors with methoctramine, activation of M1 receptors was unmasked and facilitated release.

Agonists binding nicotinic receptors elicit specific channel-opening patterns at αγ and αδ sites.

'Embryonic' muscle-type nicotinic acetylcholine receptor channels (nAChRs) bind ligands at interfaces of α- and γ- or δ-subunits. αγ and αδ sites differ in affinity, but their contributions to opening the channel have remained elusive. We compared high-resolution patch clamp currents evoked by epibatidine (Ebd), carbamylcholine (CCh) and acetylcholine (ACh). Ebd binds with 75-fold higher affinity at αγ than at αδ sites, whereas CCh and ACh prefer αδ sites. Similar short (τO1), intermediate (τO2) and long (τO3) types of opening were observed with all three agonists. τO2 openings were maximally prevalent at low Ebd concentrations, binding at αγ sites. By contrast, τO1 openings appear to be generated at αδ sites. In addition, two types of burst appeared: short bursts of an average of 0.75 ms (τB1) that should arise from the αγ site, and long bursts of 12-25 ms (τB2) in duration arising from double liganded receptors. Limited by the temporal resolution, the closings within bursts were invariant at 3 µs. Corrected for missed closings, in the case of ACh the openings within long bursts lasted 170 µs and those in short bursts about 30 µs. Blocking αδ sites with α-conotoxin M1 (CTx) eliminated both τO1 and τB2 and left only τO2 and the short τB1 bursts, as expected. Furthermore we found desensitization when the receptors bound ACh only at the αγ site. When CTx was applied to 'embryonic' mouse endplates, monoquantal current rise times were increased, and amplitude and decay time constants were reduced, as expected. Thus the αγ and αδ sites of nAChRs elicit specific channel-opening patterns.

Depolarization amplitude and Ca2+ -inflow control the time course of quantal releases at murine motor nerve terminals.

In order to test whether the time courses of quantal releases after a depolarization pulse are affected by the depolarization amplitude, time courses were measured for small depolarization pulses that elicited close to threshold releases and for large depolarizations that elicited releases approaching saturation level. Diaphragms of young mice were excised and superfused with Bretag's solution at 18 degrees C. Synaptic currents were elicited and recorded through a perfused macropatch pipette. Releases elicited by threshold depolarizations rose earlier than releases elicited by saturation depolarizations. The short delays in the rising phases of release after large depolarizations may be due to the shift of Ca(2+) currents flowing during the pulse to tail currents. After its peak, release decayed with a time constant tau. For saturation depolarizations tau was about 0.3 ms, and for threshold depolarizations tau increased up to 0.8 ms. In order to differentiate between the effects of variations in Ca(2+) inflow and in depolarization, the amplitudes of large depolarization pulses were held constant while the amount of release was depressed by halving the Ca(2+) concentration at the terminal. The time course of the lowered releases was slightly delayed while tau remained at 0.3 ms as typical for saturation depolarizations. Double pulse facilitation unexpectedly revealed a short phase of depression of release after the pulse. This depression may contribute to the rapid decay (tau) of release after large depolarizations. The dependence of tau on depolarization amplitude indicates that the final phase of the time course of release is largely controlled by the amplitude of the preceding depolarization.

Glutamatergic autoinhibition of quantal release augments the early phase of releases after a depolarization pulse.

At the crayfish neuromuscular junction, glutamatergic autoinhibition of quantal excitatory postsynaptic current (qEPSC) release is mediated by a presynaptic DL-glutamate transporter and its associated Cl- conductance. I investigated whether it also affects the time course of release. qEPSCs were recorded with a perfused macroelectrode through which depolarization pulses and D- or L-glutamate could be applied to a terminal. In order to represent the time course of release, cumulative delays of qEPSCs were determined and scaled to a common final value. At 10 degrees C, on the application of D- or L-glutamate, release increased relative to the controls especially during its first millisecond, taking the mean of 20 experiments (P < 0.01). Also, in many single experiments the respective shifts in the time courses of release were highly significant. The relative surplus of early releases decreased with time constants tau1 of 86 micros and tau2 of 0.75 ms. At 0 degrees C, in the presence of glutamate, the surplus of early delays was increased relative to the controls to a significantly greater extent and for a longer time than at 10 degrees C. The tau1 of 240 micros was almost three times larger than at 10 degrees C. Autoinhibition was inactivated in Cl(-)-free solution. In such solutions the surplus of early releases also disappeared and the shortening of early delays reverted to a lengthening. Interaction of the inhibitory autoreceptor and its associated Cl- flow with the release machinery is discussed.

Short openings in high resolution single channel recordings of mouse nicotinic receptors.

The temporal fine structure of single channel currents was studied to obtain information on how agonists open nicotinic acetylcholine receptor channels. Currents were recorded from mouse myoballs with quartz pipettes in the on-cell mode of the patch-clamp technique. With 62 kHz filter cut-off and root mean square (r.m.s.) noise levels as low as 1.45 pA at 200 mV hyperpolarization, events down to 6 micros duration could be resolved with negligible error rate. Three types of openings with mean durations of 750 micros, 89 micros and 4 micros were identified with 0.1-10 microM suberyldicholine (SubCh). The relative frequencies of the three types of openings were 84% for long, 5% for medium and 11% for short openings with 1 microM SubCh. Stability plots and single channel current amplitude comparisons suggest that the three types of openings arise from a homogenous channel population. Above 10 microM SubCh, the three types of openings could not be discerned because channel openings occurred too closely spaced and open channels were increasingly blocked. Three types of openings can be generated with a mechanistic receptor model with two unequal binding sites, short and medium openings arising from one or the other monoliganded state, and long openings from the fully liganded state of the receptor. Maximum likelihood fitting of the rate constants of this model directly to the sequence of observed open and shut times accurately predicted the main physiological properties of the receptors with 0.1 microM SubCh. However, fitting recordings with 0.1-10 microM SubCh simultaneously revealed that this model cannot reproduce the weak influence of SubCh concentration on the proportions of the three types of openings. Therefore we conclude that short and medium openings are unlikely to arise preferentially from one or the other monoliganded state of nicotinic acetylcholine receptor channels.

Both d- and l-glutamate induce transporter-mediated presynaptic autoinhibition of transmitter release.

In crayfish motor nerve terminals l-glutamate (Glu) is the excitatory transmitter and low l-Glu concentrations exert autoinhibition by inhibiting release of Glu quanta from the terminals. This autoinhibition has been shown to be mediated by binding and transport of l-Glu by Glu transporters in the presynaptic membrane. Activated transporters open an associated Cl(-) channel and inhibit release [J. Dudel & M. Schramm (2003) Eur. J. Neurosci., 18, 902-910]. The excitatory, glutamatergic synaptic transmission is specific for the l-Glu isomer. However, transporters are non-selective for the stereoisomers. It is shown here that low concentrations (5 micro m) of d- as well as l-Glu inhibit quantal release on average to 55 and 68%, respectively. The power of inhibition varies widely at different terminals but the local sensitivity to d-Glu is seen to be the same as that for l-Glu. l-Glutamate has been reported to reduce the mean amplitude of nerve terminal action currents (excitatory nerve terminal currents) by about 10%, presumably due to the opening of Cl(-) channels. Evidence is given that d-Glu also inhibits this by an average of 10% (P < 0.001), as expected if both l- and d-Glu activate a transporter-associated Cl(-) conductance. The results give further support for this novel mechanism of regulation of synaptic strength.

A receptor for presynaptic glutamatergic autoinhibition is a glutamate transporter.

Monoquantal excitatory postsynaptic currents were recorded by means of a perfused macropatch electrode from 9 to 15 micro m stretches of crayfish neuromuscular junctions. The excitatory transmitter l-glutamate superfused to a terminal inhibits quantal release by activating autoreceptors [Parnas et al. (1996) Eur. J. Neurosci., 8, 116-126]. Substances related to glutamate that do not activate glutamatergic postsynaptic channels, but are substrates of glutamate transporters, elicited analogous inhibitions, e.g. l- and d-aspartate and some other glutamate transport blockers. As expected, all transport blockers prolonged synaptic currents. Blockers that bind to the transporter receptors but are not transported did not inhibit release, but prevented inhibition by the transport substrates. It appears that autoinhibition is elicited by transport of glutamate or its analogues. Transport into cells is powered by symport of three Na+. To block the transport step electrochemically, extracellular Na+ concentration was lowered to one-quarter, but this surprisingly left the inhibition of release by glutamate unaffected, showing inhibition to be associated to a step between binding and transport. After binding a substrate, glutamate transporters open a parallel Cl- channel. Replacement of extracellular Cl- prevented Cl- current, and release inhibition by glutamate or aspartate was blocked. It is suggested that the flow of Cl- across the cell membrane, after binding a transport substrate, mediates autoinhibition. We measured a related reduction of presynaptic action potentials.

Quantal endplate currents from newborn to adult mice and the switch from embryonic to adult channel type.

Quantal endplate currents (qEPCs) were recorded extracellularly by a macropatch electrode from excised diaphragms of mice. During the first 3 days after birth, the mean rise time t(r) was 0.5 ms (0.1-0.9 peak, 20 degrees C). Double-exponential, amplitude-weighted fits of the decay discerned almost equally abundant components tau(1)' approximately equal to 6 ms and tau(2)' approximately equal to 9 ms. Beginning on day 3, on days 4 and 5 after birth both t(r) and the tau' dropped. Further decreasing slowly, adult values were reached at day 8, with t(r) approximately equal to 0.3 ms, tau(1)' approximately equal to 2 ms and a very weak tau(2)' approximately equal to 6 ms component. When compared to the kinetics of fetal channels, the tau(1)' and tau(2)' of up to 3 day qEPCs could correspond to the short and long splice variants of the fetal channel type. The tau(1)' of adult muscles of 2 ms agrees well with the burst durations of adult channels while a weak longer tau(2)' component may represent 'extrasynaptic' channels. The long t(r) of very young mice may correspond to the relatively slow rise of channel currents elicited by ACh pulses in mouse myoballs.