Single channels activated by acetylcholine in rat superior cervical ganglion.

117

SUMMARY1. The properties of acetylcholine (ACh)-activated ion channels of parasympathetic neurones from neonatal rat cardiac ganglia grown in tissue culture were examined using patch clamp recording techniques. Membrane currents evoked by ACh were mimicked by nicotine, attenuated by neuronal bungarotoxin, and unaffected by atropine, suggesting that the ACh-induced currents are mediated by nicotinic receptor activation.2. The current-voltage (I-V) relationship for whole-cell ACh-evoked currents exhibited strong inward rectification and a reversal (zero current) potential of -3 mV (NaCl outside, CsCl inside). The rectification was not alleviated by changing the main permeant cation or by removal of divalent cations from the intracellular or extracellular solutions. Unitary ACh-activated currents exhibited a linear I-V relationship with slope conductances of 32 pS in cell-attached membrane patches and 38 pS in excised membrane patches with symmetrical CsCl solutions.3. Acetylcholine-induced currents were reversibly inhibited in a dose-dependent manner by the ganglionic antagonists, mecamylamine (Kd = 37 nM) and hexamethonium (IC50 1 UtM), as well as by the neuromuscular relaxant, d-tubocurarine (Kd = 3 /M). Inhibition of ACh-evoked currents by hexamethonium could not be described by a simple blocking model for drug-receptor interaction.4. The amplitude of the ionic current through the open channel was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential upon replacement of NaCl by mannitol indicates that the neuronal nicotinic receptor channel is cation selective and the magnitude suggests a high cation to anion permeability ratio. The cation permeability (Px/PNa) followed the ionic selectivity sequence Cs+(1 06) > Na+(1P0) > Ca2+(093). Anion substitution experiments showed a relative anion permeability, PCi/pNa 1< 005.5. The nicotinic ACh-activated channels described mediate the responses of postganglionic parasympathetic neurones of the mammalian heart to vagal stimulation.

Section: Discussionmentioning

confidence: 99%

Acetylcholine‐evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia.

Fieber

Adams

1991

117

“…Mulle & Changeux (1990) (Mathie, Cull-Candy & Colquhoun, 1991). In superior cervical ganglion cells conductances of 20 and 50 pS were observed in 2-5 mm Ca2+ and 1 mM Mg2+ (Derkach, North, Selyanko & Skok, 1987 (Bormann & Matthaei, 1983), 49 pS in 0 mM Mg2+, 0 mm Ca2+ (Ifune & Steinbach, 1990), and 48 pS was reported in 2 mm Ca2+ and 0 mM Mg2+ (Sands & Barish, 1992). In retinal ganglion cells a conductance of 48 pS has been reported in 2-5 mm Ca2+ and 0 5 mM Mg2+ (Lipton, Aizenmann &Loring, 1987).…”

Section: Whole Cell Current Responsesmentioning

confidence: 99%

Heterogeneity of neuronal nicotinic acetylcholine receptors in thin slices of rat medial habenula.

Connolly

Gibb

Colquhoun

1995

1. Neuronal nicotinic acetylcholine receptors in slices of rat medial habenula were studied using patch clamp recording techniques. 2. Whole cell current responses to cytisine could be blocked by hexamethonium, as expected for nicotinic receptors. The whole cell current-voltage relations were linear at negative membrane potentials, but showed strong inward rectification when chloride currents were minimized.3. When 1 mm Ca2+ (0 mM Mg2+) was present in the external recording solution, the single channel conductances elicited by acetylcholine or nicotine in twenty patches were in the range 39-58 pS, with a mean of 47 pS. There appeared to be at least two groups of conductances. 4. In the open point amplitude distributions of three patches, the most common amplitude corresponded to 41 pS (81 % of the area). In another four patches the most common amplitude corresponded to a mean conductance of 51 pS (83 % of the area). Direct transitions between open levels were rare. 5. Channel closed times were not significantly different for the two conductance groups.However, for the four patches with predominantly 51 pS openings, the means of the distributions of open times longer than two filter rise times averaged 5'8 ms. Those patches with predominantly 41 pS openings averaged 14 ms. Also, for patches with predominantly 51 pS openings the overall mean burst length was 5-8 ms, whereas for patches with predominantly 41 pS openings it was 16-1 ms. 6. These observations suggest that 51 and 41 pS openings result from the activity of at least two, but possibly more, different receptor subtypes. We conclude that nicotinic receptors in the rat ventral medial habenula are heterogeneous.Electrophysiological, radiolabelled ligand-binding and in situ hybridization studies suggest that nicotinic acetylcholine receptors (AChRs) are widespread throughout the brain (for reviews see Deneris, Connolly, Rogers & Duvoisin, 1991;Role, 1992). These receptors have been much less well characterized than their counterparts in ganglia and at the neuromuscular junction. A recent technical development, the thin slice technique (Edwards, Konnerth, Sakmann & Takahashi, 1989), has meant that all regions of the brain can now be studied with the full range of patch-clamp techniques. Advantages of this technique are that the cells being studied can be seen directly and that many of the synaptic connections of the neurones are maintained.Within the rat brain, in situ hybridization studies have shown that the medial habenula is particularly rich in the expression of neuronal AChR subunit RNAs (Wada et al.

“…Furthermore, since there are, on average, only about 0-4 intermediate gaps per burst for ACh, the predicted value of /8 would be only about 390 s-5 if this calculation were used, a value which is probably too low to be consistent with the observed rise time of EPSCs. Derkach et al (1987) attempted a similar analysis of channels in rat superior cervical ganglion cells. They inferred rates about 4-fold slower than those estimated 745 A. MATHIE, S. G. CULL-CANDY AND D. COLQUHOUN here, largely because the shortest shut time component that they found was 150 /is compared with 26 Its that we find.…”

Section: Possible Interpretationsmentioning

confidence: 99%

Conductance and kinetic properties of single nicotinic acetylcholine receptor channels in rat sympathetic neurones.

Mathie

Cull-Candy

Colquhoun

1991

SUMMARY1. The unitary conductance of nicotinic acetylcholine (ACh) receptor channels in rat sympathetic neurones has been studied. Conductance estimates varied from 26-48 pS with a mean of 36-8 pS in 1 mM-Ca2+. The main conductance level varied from patch to patch and the presence (or absence) of additional conductance levels also varied.2. The channels showed large open channel noise and experiments with 300 mMNaCl in the patch pipette substantially increased the open channel noise. The appearance of detectable step-like transitions within this noise strongly suggested the existence of closely spaced discrete levels.3. Removal of divalent cations from the external solution increased the unitary channel conductance. Altering the main permeant ion in divalent-free solutions gave the following conductance sequence: K+ (93 pS) > Cs' (61 pS) > Na+ (51 pS) > Li+(23 pS).4. Replacement of Na+ by Cs' in the external solution considerably reduced the current evoked by ACh in whole-cell recordings and the channel-opening frequency in outside-out patches.5. The kinetic properties of channels activated by ACh and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) were also studied. At low concentrations of ACh and DMPP the gap distributions were complex and best fitted by the sum of four exponential components. Individual activations (bursts) were interrupted by the two shortest closed periods the briefer of which had time constants of 36 ,ts for ACh and 67 ,ts for DMPP.6. The distribution of burst lengths had two components for each agonist, each component making up about 50 % of the total area under the distribution. For ACh, the time constant of the longer component (12-2 ms) was similar to the decay time constant of excitatory postsynaptic potentials (EPSCs) at similar temperature and potential. For DMPP the time constant of the longer component was 17-6 ms. 8. In terms of receptor mechanism, the values of the channel opening equilibrium constant (,/oc) estimated from these numbers (ACh, 23; DMPP, 25) suggest that both agonists are efficaceous.9. DMPP is a potent blocker of the channel with an equilibrium dissociation constant (KB) of around 50 /tM and blockage gaps of around 1 ms duration. ACh also blocks the channel but with a higher KB of around 470 ,UM. 10. The equilibrium concentration-response relationship was estimated for a range of ACh concentrations by measuring the proportion of time a channel stays open (po) during the clusters of openings that occur on escape from desensitization.The p0 values for clusters at a given ACh concentration were found to be very variable, however, division of clusters into 'low po' and 'normal' allowed a plausible activation mechanism to be fitted to the data.11. A randomization test suggested that the existence of such heterogeneity is due to more than one population of clusters made up of channels with quite different open and closed times. It is not known, however, whether such differences are due to the existence of more than one channel type or to variation in the behaviour of ...