Abstract
We compared the main properties of human recombinant alpha3beta4beta3 neuronal nicotinic receptors with those of alpha3beta4 receptors, expressed in Xenopus oocytes. beta3 incorporation decreased the channel mean open time (from 5.61 to 1.14 ms, after approximate correction for missed gaps) and burst length. There was also an increase in single channel slope conductance from 28.8 picosiemens (alpha3beta4) to 46.7 picosiemens (alpha3beta4beta3; in low divalent external solution). On the other hand, the calcium permeability (determined by a reversal potential method in chloride-depleted oocytes) and the pharmacological properties of beta3-containing receptors differed little from those of alpha3beta4. The main pharmacological difference in alpha3beta4beta3 "triplet" receptors was a 3-fold decrease in the potency of lobeline relative to acetylcholine. Nevertheless, there was no change in the rank order of potency for agonists (epibatidine >> lobeline > cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, nicotine > acetylcholine > carbachol for both receptors; measured at low agonist concentrations). Sensitivity to the competitive antagonists trimetaphan (0.2-1 microM) and dihydro-beta-erythroidine (30 microM) was similar for the two combinations, with a Schild KB for trimetaphan of 76 and 66 nM on alpha3beta4 and alpha3beta4beta3, respectively. The change in single channel conductance confirms that beta3 replaces a beta4 subunit in the pentamer. The absence of pronounced differences in the pharmacological profile of the triplet receptor argues against a role for the beta3 subunit in the formation of agonist binding sites, whereas the changes in channel kinetics suggest an important effect on receptor gating. The shortening of the burst length of beta3-containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics.
Highlights
Among neuronal nicotinic subunits, 3 was long considered an “orphan” as it does not form functional recombinant receptors if expressed as a classical heteromeric combination, i.e
The shortening of the burst length of 3containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics
By a reporter mutation strategy, we showed that 3 is incorporated into functional recombinant ␣343 receptors [1]
Summary
Expression of Nicotinic Subunits cRNA in the Xenopus oocyte—cDNAs for the human ␣3, 3, and 4 (GenBankTM accession numbers Y08418, Y08417, and Y08416, respectively) containing only coding sequences and an added Kozak consensus sequence (GCCACC) immediately upstream of the start codon [4] were subcloned into the pSP64GL vector, which contains 5Ј- and 3Ј- untranslated Xenopus -globin regions [5]. All curves obtained in each experiment were fitted simultaneously by least squares (CVFIT, courtesy of David Colquhoun, UCL; see www.ucl.ac.uk/Pharmacology/ dcpr95.html) with power functions constrained to be parallel (these are the equivalent of fitting with a Hill equation in which the maximum is constrained to be very large with respect to the measured responses). In a manner similar to that employed for the agonist potency ratios, the dose ratio r produced by the antagonist was measured for each experiment by fitting to the partial concentration-response curves power functions constrained to be parallel. Calcium Permeability—We measured the effect of different calcium concentrations on the reversal potential of the current induced by 2-s voltage ramps from Ϫ40 to 40 mV holding potential Such ramps were applied in the depolarizing direction and in the hyperpolarizing direction both in control conditions (leak) and in the presence of ACh (test) once the agonist current was steady.
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