1. The activation of elementary calcium release events ('puffs') and their co-ordination to generate calcium waves was studied in Xenopus oocytes by confocal linescan imaging together with photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor. 2. Weak photolysis flashes evoked no responses or isolated calcium puffs, whereas flashes of increasing strength evoked more frequent puffs, often occurring in flurries as abortive waves, and then a near-simultaneous calcium liberation originating at multiple sites. The numbers of sites activated increased initially as about the fourth power of photoreleased [InsP3]. 3. Following repeated, identical photolysis flashes, puffs arose after stochastically varying latencies of a few hundred milliseconds to several seconds. The cumulative number of events initially increased as about the third power of time. No rise in free [Ca2+] was detected preceding the puffs, suggesting that this co-operativity arises through binding of multiple InsP3 molecules, rather than through calcium feedback. 4. The mean latency to onset of calcium liberation shortened as about the square of the flash strength, and the dispersion in latencies between events reduced correspondingly. 5. Weak stimuli often evoked coupled puffs involving adjacent sites, and stronger flashes evoked saltatory calcium waves, propagating with non-constant velocity. During waves, [Ca2+] rose slowly between puff sites, but more abruptly at active sites following an initial diffusive rise in calcium. 6. Initial rates of rise of local [Ca2+] at release sites were similar during puffs and release induced by much (> 10-fold) greater [InsP3]. In contrast, macroscopic calcium measurements averaged over the scan line showed a graded dependence of rate of calcium liberation upon [InsP3], due to recruitment of additional sites and decreasing dispersion in activation latencies. 7. We conclude that the initiation of calcium liberation depends co-operatively upon [InsP3] whereas the subsequent regenerative increase in calcium flux depends upon local calcium feedback and is largely independent of [InsP3]. Wave propagation is consistent with the diffusive spread of calcium evoking regenerative liberation at heterogeneous discrete sites, the sensitivity of which is primed by InsP3.

ATP has recently been identified as a fast neurotransmitter in both the central and peripheral nervous systems. Several studies have suggested that ATP can also affect the release of classical neurotransmitters, including acetylcholine with which it is co-released. We have searched for ATP receptors on a cholinergic presynaptic nerve terminal using the calyx-type synapse of the chicken ciliary ganglion. ATP was pulsed onto the terminals under voltage clamp and induced a short latency cation current that exhibited inward rectification and marked desensitization. This current was not seen with adenosine but was mimicked by several sterically restricted ATP analogs and was blocked by suramin. ATPactivated single ion channels exhibited prominent flickering and had a conductance of -17 pS. Our results demonstrate a ligand-gated P2x-like purinergic receptor on a cholinergic presynaptic nerve terminal.ATP is a prime candidate as a transmitter release modulator in cholinergic nerve terminals and can result in both up-and down-regulation of synaptic transmission. Although some of these actions are due to the breakdown product adenosine (1-3), ATP itself can also affect transmitter release (4, 5). ATP is known to activate nonselective cation channels on neurons and muscle fibers (6-8) primarily via the P2X purinergic receptor subtype, but there is currently no direct evidence localizing these channels on presynaptic nerve terminals. We have previously demonstrated that it is possible to record whole-cell and single-channel currents from the extensive calyx-type cholinergic presynaptic nerve terminals of the chicken ciliary ganglion (9, 10). We have used this preparation to test for the presence of presynaptic ATP-gated ion channels by treating the calyces with purinergic agonists and blockers. Some of these results have been presented in abstract form (11,12). EXPERIMENTAL PROCEDURESPreparation of Calyx and Neuron. Calyx nerve terminals were isolated as described (13) with minor modifications (10). Briefly, ciliary ganglia were dissected from 15-day-old chicken embryos and were incubated in modified Eagle's medium (MEM) containing 0.74 mg of collagenase IV per ml (Worthington, Freehold, NJ), 12.3 mg of dispase per ml (Boehringer Mannheim), 800 units of hyaluronidase per ml (Worthington), and 0.07 mg of trypsin inhibitor II per ml (Sigma) for 2-3 hr at 37°C in 5% CO,/95% air. The digestion was terminated by removing ganglia from the enzyme solution and washing with MEM. The ganglia were triturated in the presence of the vital dye 4-(4-diethylaminostyl) N-methyl-pyridinium (4-Di-2-ASP; Molecular Probes, Eugene, OR) at 80 nM and the cell suspension was allowed to adhere on a glass coverslip recordingThe 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.chamber. Experiments were carried out at room temperature (22-25°C).Solutions. The standard internal solution for ...

SUMMARY1. A large-conductance Cl-channel was characterized in cell-free membrane patches from the rabbit longitudinal colonic smooth muscle using the patch clamp technique. In addition, the regulation of these channels by neurokinin-1 (NK-1) receptor agonists and G proteins was studied.2. No spontaneous channel activity was observed in cell-attached patches at the cell resting potential, or in excised patches at pipette potentials (Vp) between -20 and 20 mV. In excised patches, channel activity could be induced in thirty-six out of ninety-six patches by holding the patch at Vp values more negative than -60 mV or more positive than 60 mV. Once induced, the channel showed a bell-shaped voltage activation curve in high symmetric [Cl-], with maximal open probability between 20 and -5 mV. Varying cytosolic calcium concentration ([Ca2+]) between 5 x 10-8 M and 1P0 mm had no effect on the voltage activation of the channel.3. In inside-out and outside-out patches, when pipette and bath solutions contained equal [Cl-] (130 mM), the anion channel showed a linear current-voltage (I-V) relationship between -60 and 60 mV with a slope conductance of 309 + 20 pS (n = 13). Reversal potential measurements indicated that the channel was selective for Cl-over Na+ and K+ (PCl/PNa = 6: 1).4. Channel openings from the closed state to the full open state as well as transitions through smaller conductance states were observed. The smallest detectable substate had a conductance of 15-6 pS. Based on the similarities in selectivity and linearity of the I-V curve of the smaller conductances with the full open state, and kinetic analysis of channel activity, it is concluded that the large conductance channel is composed of multiple substates which can either open and close independently, or simultaneously via a main gate.5. The stilbene derivative diiso-thiocyanato-stilbene-disulphonic acid (DIDS) and the diphenylamine-2-carboxylate analogue 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) caused a dose-dependent, reversible flicker block of the small conductance and significantly reduced the macroscopic current flow through the channel.6. In quiescent outside-out patches, when the pipette contained a 140 mM-CsCl MS 9458 36. P. SUN AND OTHERS solution with 10-6 M-CaCl2, 12 mM-MgCl2 and 1 mM-GTP, and the bath contained Ringer solution, addition of the NK-1 receptor antagonists substance P methylester resulted in activation of the full conductance state and of smaller substates.7. In inside-out patches, when the NK-1 agonist (10`1 M) was added to the pipette solution (Ringer) and the bath contained 100 mM-KCl with 10-6 M-CaCl2, 1P2 mM-MgCl2, 30 mM-TEA' and 1 mM-GTP, channel activity in the form of substates was seen in eight out of ten patches. The full open state was only seen after additional voltage induction. 8. In quiescent inside-out patches at a t' of 20 mV, when the pipette contained 90 mM-N-methyl glucosamine chloride (NMGCl), 30 mM-TEACl, 1 mm-CaCl2 and 1P2 mM-MgCl2, and the bath was a 130 mM-NaCl solution with 1 mm-CaCl2, additio...

Because of the high intracellular Cl- concentration ([Cl-]i) in gastrointestinal smooth muscle, receptor-mediated opening of Cl- channels at the cell resting potential could represent a plausible mechanism for initial receptor-mediated cell depolarization. To test this hypothesis, we characterized activation of large-conductance Cl- channels by the neurokinin-1 (NK-1) receptor agonist [Sar9,Met(O2)11]-substance P, by specific second messengers, and by direct G protein activation in myocytes isolated from the rabbit colon longitudinal muscle layer. In excised inside-out patches, large-conductance ion channels selective for Cl- over Na+ could be induced by holding the patch at pipette potentials values > 60 mV. The channel showed multiple smaller conductance states (< or = 20) but could open and close via a main gate. When the channel was fully open, its slope conductance was 300 pS, with substates as small as 15 pS, comparable to the predominant conductance observed in cell-attached patches. The voltage-activation profile for full conductance was bell-shaped with maximal open probability (Po) for channel opening of approximately 0 mV. In cell-attached patches, addition of the NK-1 agonist to pipette solution activated a channel that corresponded to a subconductance state of the maxi Cl- channel. The voltage-activation profile for this subconductance state showed a maximal Po value for membrane potentials of approximately 0 mV, with rapid inactivation at more positive and partial inactivation at more negative membrane potentials. In excised inside-out patches, both the full and smaller conductance states of the Cl- channel were activated by the nonhydrolyzable guanosine triphosphate analogue guanosine 5'-O-(3-thiotriphosphate) and inhibited by pertussis toxin (PTX), whereas [Ca2+]i increased channel activity only in concentrations > 1 mM. In cell-attached patches, addition of different Ca2+ ionophores resulted in channel activation in 10% of cells, and activators of protein kinase A or protein kinase C had no effect. These findings are consistent with the hypothesis of a possible role of G protein-coupled Cl- channels in receptor-mediated initial cell depolarization in longitudinal colonic smooth muscle.