Kinetics and Equilibria at Nicotinic Receptors in Electrophorus Electricus and Raia Erinacea Electroplaques

Author: Sheridan, Robert Edward, Jr.

Year: 1978

Degree: Dissertation (Ph.D.)

Advisor: Lester, Henry A.

Committee Member: Unknown, Unknown

Option: Neurobiology; Biochemistry

DOI: 10.7907/dvbb-4m95

Abstract

This study utilizes relaxation kinetics and equilibrium conductance to measure the response of the nicotinic receptor to cholinergic agonists. Agonist-induced conductance, postsynaptic currents (PSC's), and voltage-jump relaxations are studied in electroplaques of Electrophorus electricus. Agonist-induced conductance is instantaneously ohmic at voltages more negative than -30 mV. In bath-applied acetylcholine (ACh), the dose vs. conductance relation is sigmoid. At 15°, the apparent dissociation constant for ACh decreases e-fold for every 87 mV of hyperpolarization, hence agonist-induced conductance increases as the electroplaque is hyperpolarized. In other experiments, presynaptic terminals are stimulated to produce PSC's. Peak PSC changes linearly with membrane voltages more negative than -30 mV. The estimated reversal potential for all the above agonist-induced currants is about +10 mV. After the peak, PSC's decay with a single exponential rate, ∝. At 15°, ∝ equals 1.2 msec-1 at 0 mv and decreases e-fold for every 86 mV of hyperpolarization. In still another series of experiments, an instantaneous jump in membrane voltage causes agonist-induced conductance to relax to a new equilibrium along a simple exponential time course. The rate constant, 1/τ, for such relaxations varies with the nature of the agonist, its concentration, and the final membrane voltage. This relaxation rate is interpreted as the sum of the closing rate of receptors, ∝, and a first-order, voltage-independent rate constant tor receptor opening. As expected from this interpretation: (a) 1/τ approaches ∝ at low ACh concentrations, (b) 1/τ increases linearly with agonist concentration, and (c) 1/τ is unaffected by blockade of receptors with ∝-bungarotoxin. Several kinetic models of the nicotinic receptor are tested. The one which best fits the data assumes: (a) that the open state of the receptor forms as the receptor binds the second in a series of two agonist molecules, (b) that this process constitutes the rate-limiting step in receptor activation, and (c) that dissociation of either agonist molecule closes the receptor. In ACh, the rate-limiting step proceeds with an opening rate or 107 M-1sec-1 and a closing rate of 102 to 103 sec-1.

Agonist-induced conductance and postsynaptic currents are also studied in electroplaques or Raia erinacea. In Raia electroplaques, delayed rectification prevents 11easurement of conductance at voltages more positive than -.50 mV. At voltages more negative than -50 mV, bath-applied carbachol (3 to 9 µM) produces a steady-state conductance which is independent of membrane voltage. In ether experiments, PSC's are produced by electrical stimulation of presynaptic nerves. Peak PSC varies linearly with membrane voltage. At 20°, the extrapolated reversal potential for all the above agonist-induced currents is about 0 mV. PSC's decay exponentially after the peak. The rate constant of the decay, ∝, does not vary with membrane voltage and equals 0.23 msec-1 at 20°. This rate constant does increase with temperature and has a Q10 of 1.95. D-tubocurarine reduces the peak PSC but does not change the decay rate. Neostigmine reduces the decay rate but does not change the peak PSC. These results imply that the opening and closing rates of the nicotinic receptor are voltage insensitive in Raia electroplaques.

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