A B S T R A C T This study used messenger RNA encoding each subunit (a, fl, % and 6) of the nicotinic acetylcholine (ACh) receptor from mouse BC3H-1 cells and from Torpedo electric organ. The mRNA was synthesized in vitro by transcription with SP6 polymerase from cDNA clones. All 16 possible combinations that include one mRNA for each of a, fl, % and ~ were injected into oocytes. After allowing 2-8 d for translation and assembly, we assayed each oocyte for (a) receptor assembly, measured by the binding of [12sI]a-bungarotoxin to the oocyte surface, and (b) ACh-induced conductance, measured under voltage clamp at various membrane potentials. All combinations yielded detectable assembly (30-fold range among different combinations) and ACh-induced conductances (>l,000-fold range at 1 #M). On double-logarithmic coordinates, the dose-response relations all had a slope near 2 for low concentrations of ACh. Data were corrected for variations in efficiency of translation among identically injected oocytes by expressing ACh-induced conductance per femtomole of a-bungarotoxin-binding sites. Five combinations were tested for dtubocurarine inhibition by the dose-ratio method; the apparent dissociation constant ranged from 0.08 to 0.27 #M. Matched responses and geometric means are used for describing the effects of changing a particular subunit (mouse vs. Torpedo) while maintaining the identity of the other subunits. A dramatic subunit-specific effect is that of the fl subunit on voltage sensitivity of the response: gACh(--90 mV)/gAch(+30 mV) is always at least 1, but this ratio increases by an average of 3.5-fold if fl~a replaces fiT. Also, combinations including "YT or 6M usually produce greater receptor assembly than combinations including the homologous subunit from the other species. Finally, EACh is defined as the concentration of ACh inducing 1 #S/fmol at -60 mV; EACh is consistently lower for am. We conclude that receptor assembly, voltage sensitivity, and EACh are governed by different properties.

Responses in the frog glossopharyngeal nerve inducd by electrical stimulation of the tongue were compared with those induced by chemical stimuli under various conditions. (a) Anodal stimulation induced much larger responses than cathodal stimulation, and anodal stimulation of the tongue adapted to 5 mM MgCI2 produced much larger responses than stimulation with the tongue adapted to 10 mM NaCI at equal current intensities, as chemical stimulation with MgCI2 produced much larger responses than stimulation with NaCI at equal concentration. (b) The enhansive and suppressive effects of 8-anilino-l-naphthalenesulfonate, NiCI2, and uranyl acetate on the responses to anodal current were similar to those on the responses to chemical stimulation. (c) Anodal stimulation of the tongue adapted to 50 mM CaCI2 resulted in a large response, whereas application of 1 M CaC12 to the tongue adapted to 50 mM CaCI2 produced only a small response. This, together with theoretical considerations, suggested that the accumulation of salts on the tongue surface is not the cause of the generation of the response to anodal current. (d) Cathodal current suppressed the responses induced by 1 mM CaCI2, 0.3 M ethanol, and distilled water. (e) The addition of EGTA or Ca-channel blockers (CdC12 and verapamil) to the perfusing solution for the lingual artery reversibly suppressed both the responses to chemical stimulus (NaCI) and to anodal current with 10 mM NaCI. (f) We assume from the results obtained that electrical current from the microvillus membrane of a taste cell to the synaptic area supplied by anodal stimulation or induced by chemical stimulation activates the voltage-dependent Ca channel at the synaptic area.

1. The excitable properties of branched cells were measured using a combination of voltage-clamp and frequency-domain techniques. Point impedance functions from either the soma or growth cone of NG-108 cells were curve fitted with a reduced cable model at different membrane potentials to establish kinetic parameters. 2. Transfer impedance functions between the soma and growth cone were measured and simulated with a morphologically determined model. In these experiments the membrane potential was controlled by a single-electrode voltage clamp thus allowing an estimate of transfer functions for any arbitrary input, such as a single synaptic current for differing degrees of tonic synaptic drive. Furthermore, the integration of different regional inputs was evaluated based on the transfer functions between different locations on an individual cell. 3. The activation of an outward steady-state current leads to resonating impedance functions that were used to evaluate the kinetic properties of ionic channels in different regions of branched excitable cells. For simple branching patterns the point and transfer impedances show lower resonant frequencies for active growth cones compared with active somas. 4. More complex branching patterns showed the unexpected result that the voltage-dependent resonant frequency was higher for the growth cone recording than the soma. The presence of a higher resonant frequency when the growth cone is activated does not require more rapid kinetics of the active potassium conductance, since the time constant of the active conductance can be the same in the growth cone and the soma membrane. 5. In conclusion, the resonant frequencies, as well as all other aspects of the impedance functions, are complicated interactions of the detailed branching patterns and active conductances. In general, these interactions are not predictable from a passive electrotonic analysis, especially when the voltage-dependent conductances are distributed throughout the dendritic tree.

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1. Impulse response functions were determined from complex point impedance and transfer functions from cultured NG-108 cells to simulate the propagation of a synaptic potential in response to the release of transmitter. In general, the flow of synaptic current has a much shorter duration than the normal membrane time constant, thereby making the use of impulse response functions useful approximations to synaptic events. 2. The resonance observed during the activation of the potassium conductance was reflected in the impulse response function as a pronounced damped oscillation. A comparison of the impulse response functions calculated from point impedance and transfer functions showed similar results for current injections in the growth cone. 3. In addition to the resonance effects of the voltage-dependent conductances on transfer and impulse response functions due principally to the activation of conductances for outward currents, transfer functions were measured during the activation of a steady-state negative conductance. Under these conditions the phase function approaches 180 degrees, indicating that the voltage response is out of phase with the current. 4. In the steady state, the effect of a negative conductance is to algebraically add to the positive conductances and generally decrease the absolute conductance unless there is a net negative current. The decreased conductance enhances the impulse response and the DC space constant, thus leading to a better propagation of slow potentials. This effect can be seen as a decrease in the electrotonic length, L, with intermediate depolarizations. At large depolarizations the steady-state activation of the K conductance generally dominates and leads to a greatly increased electrotonic length. 5. Both the net conductances and the associated kinetics play a role in shaping the potential changes during a synaptic current. This is especially critical if there is a net negative steady-state conductance. Under these conditions there is a surprising reduction in the impulse response function. 6. Thus, during a subthreshold activation of the voltage-dependent negative conductances, the observable synaptic potentials would be either large potential responses due to an apparent increase in the impedance (algebraic summation of positive and negative conductances with a net positive conductance) or a minimal response because of the phasic cancellation due to a net negative conductance. The latter condition could exist near the synaptic reversal potential due to a large synaptic drive and would appear experimentally as a form of inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)