Three types of acetylcholine-induced single channel currents in clonal rat pheochromocytoma cells

102

SUMMARY1. Single-channel currents were recorded from Xenopus oocytes which had been injected with complementary RNAs (cRNAs) for the neuronal nicotinic acetylcholine receptor subunits ax3 and f4. The co-expression of ax3 aI. 1/84 gave rise to three different channel types with conductances of 22, 18 and 13 pS.2. The activity arising from the expression of these two subunits was compared with that observed when the a3 subunit was co-expressed with an alternative 18-subunit, 182. The az3fl4-receptors differed from a3fl2-receptors in conductance, open times and most notably, in burst kinetics. The association of the f,4-subunit with the a3-subunit results in receptors which have a high probability of re-opening after closing, yielding protracted bursts of activity not observed when the a3-subunit is associated with the f82-subunit.3. All three a3,f4-channel types had similar burst kinetics. However, the 13 pS conductance channels showed an additional long-lived open state to varying degrees when clusters of activity within an individual record were examined.

Section: R L Papke and S F Heinemann Discussionsupporting

confidence: 50%

The role of the beta 4‐subunit in determining the kinetic properties of rat neuronal nicotinic acetylcholine alpha 3‐receptors.

Papke

Heinemann

1991

102

“…It is, however, rather lower than the value of 55-60 pS found at the adult mammalian endplate under similar conditions (20-25°C, 1-5 mM-external Ca2+ (Brehm & Kullberg, 1987;Mulrine & Ogden, 1989)), though it is similar to the value of 32-5 pS, found under these conditions, for the embryonictype nicotinic receptor of rat myoballs (Jaramillo & Schuetze, 1988). There are, however, a number of studies that have reported quite different (usually smaller) conductance values for neuronal nicotinic receptors, either as the main conductance value, or as one or more of a number of multiple-conductance states (Bormann & Matthaei, 1983;Moss et al 1989;Papke et al 1989;Mulle & Changeux, 1990). Particularly surprising is the observation of Papke et al (1989), that expression of the o3 and ,f2 subunits in Xenopus oocytes gives single channels with a conductance around 15 pS.…”

Section: Variability Of Conductancesupporting

confidence: 59%

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 ...

“…possessed a subpopulation of small-amplitude events: 35 pS compared with 409 pS. This may reflect variable conductance states resembling those described for ACh channels in PC12 cells, a tumour cell line derived from chromaffin cells (Bormann & Matthaei, 1983) or it may simply mean that the amplitude of the unitary conductance channel varies from channel to channel (i.e. larger standard deviation).…”

Section: S G Cull-candy a Mathie And D A Powis Channel Kineticsmentioning

confidence: 87%

Acetylcholine receptor channels and their block by clonidine in cultured bovine chromaffin cells.

Cull-Candy

Mathie

Powis

1988

SUMMARY1. Acetylcholine (10-50 /M) applied to bovine chromaffin cells under whole-cell voltage clamp produced an inward current and an increase in the noise level of the current trace. At concentrations > 20 fM the ACh current desensitized with time. Spectral analysis of the ACh-induced increase in current noise showed that it could be fitted by a single Lorentzian component with a time constant, Tnoise = 11 + 0-9 ms (Vm =-80 mV).2. Clonidine (2-30 /SM) markedly reduced the size of the ACh-induced whole-cell current, and altered the shape of the noise spectrum. In the presence of clonidine, ACh noise spectra were fitted by two Lorentzian components with time constants which varied with clonidine concentration. The single-channel conductance from noise (y = 24 pS) was unaltered by clonidine.3. The reduction in the size of the whole-cell ACh current, produced by clonidine, was not mimicked by adrenaline (at concentrations up to 60 uM). GABA-induced whole-cell currents and spectra of GABA noise were also unchanged suggesting that the effect of clonidine was specific for the ACh receptor channel.4. When applied intracellularly (from the patch pipette) clonidine had no apparent influence on the whole-cell ACh current. Furthermore, when clonidine was perfused over the cell surface at a concentration sufficient to block the whole-cell ACh response, single ACh channels could still be recorded under the patch-pipette tip (i.e. in cell-attached patches, not exposed to clonidine).5. Clonidine block showed little voltage dependence; the peak ACh current remained linearly dependent on clamp potential. In addition, the fast and slow time constants derived from ACh noise spectra in the presence of clonidine did not show the sort of dependence on antagonist concentration expected for simple channel block; this suggests that clonidine has a complex blocking action.6. Single ACh channels recorded in outside-out patches had a conductance of y = 39 + 0 7 pS, although a subpopulation of smaller and larger events also occurred in some patches. The mean single-channel conductance in outside-out patches was unaltered by clonidine.