Thermodynamic Parameters of End-plate Channel Blockade1

The reaction kinetics for drug blockade of postsynaptic channels at the mouse neuromuscular junction were studied. The activation energies associated with the onward and off-rate constants, from a sequential model, were measured for procaine, scopolamine, octanol, and heptanol, and from these the transition state thermodynamic parameters of free energy (AG$), enthalpy (AHI), and entropy (ASS) of activation were determined. All agents showed positive entropies of activation for the channel blocking rate constant kp, with the values for the alcohols significantly greater than those for the two positively charged local anesthetics. No significant differences in the activation energies for the off-rate constant ke2 were observed between any of the agents. The magnitude of k-, decreased as the hydrophobicity of the agent increased. The large activation energies measured for k2 and kp2 (in excess of 10 kcal/mol) are higher than expected for simple rate-limiting diffusion, and the possibility exists that channel blockade involves conformational changes in a protein segment, induced by a hydrophobic interaction between agent and intrachannel site or sites. The large entropy increase observed in the blocking step is indicative of the agent replacing structured waters of hydration near a hydrophobic region of a protein. A number of diverse agents cause the decay of miniature end-plate currents (MEPCs) to be divided into two exponential components. Considerable evidence has been presented which indicates that a sequential model can account for the biphasic MEPCs observed. The faster than normal component is due to “blockade” or “plugging” by the agent of ionic channels opened as a consequence of the combination of acetylcholine (ACh) with receptors, whereas the slower than normal component results from oscillation of the channel between open and “blocked” states (Adams, 1976, 1977; Pennefather and Quastel, 1980). The onand off-rate constants, kp and ke2, for “blocking” of end-plate channels by positively charged agents (but not agents presumed to act in the neutral form) are sensitive to transmembrane potential (Pennefather and Quastel, 1980) and to raised concentration of Mg”+ or Ca2+ ions (McLarnon and Quastel, 1983). In the present study, we have examined the tempera’ This work was supported by grants from the Muscular Dystrophy Association of Canada and the British Columbia Health Care Research Foundation. We thank Dr. J. Benbasat, Department of Biochemistry, The University of British Columbia, for helpful comments regarding the manuscript. 2 To whom correspondence should be addressed. ture dependence of k2 and he2 for a group of agents which cause biphasic MEPCs, namely, the aliphatic alcohols noctanol and n-heptanol, the local anesthetic procaine, and scopolamine, which, at the concentration employed (100 yM), acts like a local anesthetic on end-plate channels. Analysis, in terms of the thermodynamic parameters of rate theory, indicates that the “blocking” of end-plate channels by these agents is determined by a rate-limiting entropy-driven step, indicating hydrophobic interaction between agent and site(s). The measured activation energies for channel blockade by all agents (in excess of 11 kcal/mol) were higher than expected for a diffusion-limited process, and for the two alcohols they were significantly greater than for the two positively charged agents. The activation energies for kp2 were essentially the same for all of the agents, with the magnitude of kwp dependent upon the hydrophobicity of the agent. The large activation energies associated with k2 and k-, could be due to the agents causing conformational changes in an interfacial hydrophobic region of a protein molecule. Materials and Methods The preparation used was the mouse diaphragm; MEPCs recorded from voltage-clamped end-plates were recorded in the temperature range from 8°C to 25°C and