Ogden et al. reply

TIPS - September

1987 [Vol. 81

335 signal transmission can be blocked. This distinction, perhaps subtle to the reader, would have demonstrably dramatic effects if during surgery the local anesthetic used operates by the channelblocking mechanism.

A case for signal transmission blockage with ACh antagonists

Voltage-dependent inhibition of the acetylcholine receptor In a recent Letter to the Editor’ Dr Ogden and colleagues indicated that their elegant kinetic data obtained from single-channel current recordings support a mechanism in which antagonists to the acetylcholine receptor act solely by blocking the open channel (blocking mechanism) rather than the mechanism that we proposed2, in which the inhibitors, local anesthetics like procaine, or agonists at high concentrations bind to a pre-existing inhibitory site on the receptor (isosteric mechanism). The mechanisms are similar in that in both the open channel can be blocked. Therefore, in both mechanisms the time the channel is unblocked, and the frequency with which the blockages occur, depend on inhibitor concentration. Two published measurements are inconsistent with the channel-blocking mechanism. l In the isosteric mechanism two reaction pathways lead to the closed-channel form of the receptor, when the channel is blocked or not blocked by the inhibitor. In contrast, in the blocking mechanism a single reaction pathway leads to the closed-channel form, when the channel is not blocked by the inhibitor. This requires that the time integral of current during the open-channel state is independent of inhibitor concentration3. In measurements made by Neher4, similar to those done lay Ogden and Colquhoun but with the inhibitor QX222 (a lignoCaine derivative), this requirement is not met. l Dr Ogden and his colleagues state in their Letter that the equilibrium inhibition for both mechanisms is described by the same equation. This is only true at agonist concentrations at which the sites controlling channel opening are fully occupied. At lower agonist concentrations the two mechanisms differ, since in the blocking mechanism the inhibitor can bind only to the open-channel form. The blocking mechanism I-,.:I-:c*.. requires, therefure, iii& ~I,~~~~VILCI binding, as measured by the apparent dissociation constant KR,

is directly proportional to the concentration of the o en-channel P form of the receptor. Chemicdi kinetic measurements indicate, however, that as the acetylcholine concentration is increased so that the concentration of the open channel form increases by a factor of -7, I& remains for two different inhibitors constant5. In a sense, both mechanisms involve channel blockage. In the channel-blocking mechanism, the channel opens and signal transmission occurs before the channel is blocked, whereas in the isosteric mechanism the inhibitor is in place, the open-channel form of the receptor cannot function, and

Ogden et al. reply Hess and Udgaonkar make two points. The first is that block of acetylcholine-activated channels by QX222 does not behave in the manner expected for simple openchannel block. Although this is true for QX222, and for many other open channel blockers too, we provided evidence that the agonist dicholine suberate (suberyl dicholine) appears to act as a simple open-channel blocker’. However, we are concerned with acetylcholine and we wished to point out that our studies did not (and, by their nature, couid not) provide evidence for anything other than simple block by acetylcholine and cannot be taken to support a more general mechanism. Their second point concerns their evidence for a form of block that does not require the channel to be open and which, at negative membrane potentials, is postulated to result in self-inhibition by acetylcholine so powerful that only about 1% of channels will be opened by acetylcholine. We apologize for simplifying, for the sake of brevity, the theoretical argument. However, our reaction to this proposal is not primarily a theore:ical one, but tC pOi9t fi?f, Z!5 h our original letter, that it is an experimental observation (in frog

GEORGE JAYANT

I’. HESS

AND

B. UDGAONRAR*

Secretion of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 24853 USA and *Department of Biochemistry, Stanford University, Stanford CA 943OS, USA.

References 1 Ogden, D. C., C6lquhoun. D. and Gibbs, A. J. (1987) Trends Pharmlrcol. Sci. 8. 294295 2 Udgaonkar, J. B. and Hess, G. P. (1987) Trends Pharmacoi. Sci. 8, 190-192 3 Ogden, D. C. and Colquhoun, D. (1985) Proc. R. Sot. London Ser. B. 225,329-355 4 Neher, E. (1983) 1. Physiol. (London) 339, 663-678 5 Karpen,J. W. and Hess, G. P. (1986) Biochemistry 25‘1777-1785

muscle) that application of high acetylcholine concentrations can open almost all of the channels present, and that such a large degree of self-block is not compatible with what is known about the margin of safety for neuromuscular transmission. It seems likely, therefore, that there must be a difference between the behaviour of receptors from muscle and from electric organs, or, possibly, that the use of arginine as a sodium substitute to produce a potential difference across the vesicle membrane2 has caused some problem (arginine is known itself to produce a voltagedependent block of acetylcholineactivated channels in frog and rat muscle3. DAVID

OGDEN,

DAVID .ND

COLQUHOUN’

ALASDAIR

GIBB*

of Pharmacology, King’s College London, UK, and ‘MRC Receptor Mechanisms Research Group, Department of _Fhannacology, University College L.ondon, UK. Department

References 1 Ogden, D. C. and Colquhoun,

D. (1985) Proc. R. Sot. London Ser. B. 8225,32%355

2 Takeyasu, K. , Shiono, S., J. B., Fujita, N. and Hess, Biochemistry 25,177@-1776 __ . _: 3 sancnez, 1, n., Dani, j. A., and Hille. B. (1956) J. Gen. 985-1001

Udgaonkar, C. P. (1986) &men, D. Physiol. 87,

@ 1987, ElsevierPublications,Cambridge 0165 - 614?/87lW2.W