Activation and block of the adult muscle-type nicotinic receptor by physostigmine: single-channel studies.

The plant-derived acetylcholinesterase inhibitor physostigmine has previously been shown to act on the nicotinic acetylcholine receptor (nAChR) causing either direct activation or potentiation of currents elicited by low concentrations of nicotinic agonists, or, at higher concentrations, channel block. We examined mouse adult-type muscle nAChR activation by physostigmine and found that channel activation by physostigmine exhibits many characteristics common with channel activity elicited by nicotinic agonists. Single-channel conductance was indistinguishable, and mutants known to slow channel closing in the presence of nicotinic agonists had a similar effect in the presence of physostigmine. However, physostigmine is a very inefficacious agonist. The presence of physostigmine did not alter the effective opening rate for a subsaturating dosage of carbachol, suggesting that physostigmine does not interact with the nicotinic agonist binding site. Mutations to a residue (alphaLys125) previously identified as part of the putative binding site for physostigmine reduced the duration of openings elicited by physostigmine, but the effects were generally small and, in most cases, nonsignificant. At higher concentrations, physostigmine blocked channel activity. Block manifested as a reduction in the mean open time and the emergence of a closed state, with a mean duration of 3 to 7 ms. The properties of block were consistent with two equivalent blocking sites per receptor with microscopic binding and unbinding rate constants for physostigmine of 20 microM(-1) s(-1) and 450 s(-1) (K(D) = 23 microM). These observations indicate that physostigmine is able to activate muscle nAChR by interacting with a site other than the nicotinic ligand binding site.

The role of loop F residues in determining differential d-tubocurarine potencies in mouse and human 5-hydroxytryptamine 3A receptors.

The competitive antagonist d-tubocurarine (curare) has greater potency at mouse than at human 5-hydroxytryptamine 3A (5-HT3A) receptors, despite 84% amino acid sequence identity between the receptors. Within the ligand binding domain of this receptor are six loops (A-F). A previous report demonstrated that loop C of the 5-HT3A receptor contributed to differential potency between the receptors [Hope, A. G. et al. (1999) Mol. Pharmacol. 55, 1037-1043]. The present study tested the hypothesis that loop F plays a significant role in conferring interspecies curare potency differences. Wild-type, chimeric, and point mutant 5-HT3A receptors were expressed in Xenopus oocytes, and two-electrode voltage clamp electrophysiological recordings were performed. Our data suggest that loops C and F contribute to curare potency, given that the curare IC50's (concentration of drug that produces 50% inhibition of the response) for chimeric human receptors with substitutions of mouse residues in loop C (40.07 +/- 2.52 nM) or loop F (131.8 +/- 5.95 nM) were intermediate between those for the mouse (12.99 +/- 0.77 nM) and human (1817 +/- 92.36 nM) wild-type receptors. Two human point mutant receptors containing mouse receptor substitutions in loop F (H-K195E or H-V202I) had significantly lower curare IC50's than that of the human receptor. The human double mutant receptor, H-K195E,V202I, had the same curare IC50 (133.8 +/- 6.38 nM) as that of the human receptor containing all six loop F mouse substitutions. These results demonstrate that two loop F residues make a significant contribution in determining curare potency at the 5-HT3A receptor.

Age-related retinal degeneration in animal models of aging: possible involvement of taurine deficiency and oxidative stress.

There is strong evidence that the retina degenerates with age. Electroretinogram deficits and photoreceptor cell death and structural abnormalities have been observed in both animal and human studies of aging. The mechanism behind this phenomenon is a very interesting area for scientific and medical study. Current data support the link between retinal degeneration and increased oxidative stress. Taurine is a free amino acid found in high millimolar concentrations in the retina, and age-related deficiency in retinal levels of taurine may contribute to the retinal degeneration associated with age. Taurine acts as an antioxidant and taurine replenishment is known to alleviate oxidative stress in the retina. Thus taurine supplementation may be useful in the treatment of age-related retinal dysfunction.

Taurine: evidence of physiological function in the retina.

Taurine is a free amino acid found in high millimolar concentrations in mammalian tissue and is particularly abundant in the retina. Mammals synthesize taurine endogenously with varying abilities, with some species more dependent on dietary sources of taurine than others. Human children appear to be more dependent on dietary taurine than adults. Specifically, it has been established that visual dysfunction in both human and animal subjects results from taurine deficiency. Moreover, the deficiency is reversed with simple nutritional supplementation with taurine. The data suggest that taurine is an important neurochemical factor in the visual system. However, the exact function or functions of taurine in the retina are still unresolved despite continuing scientific study. Nevertheless, the importance of taurine in the retina is implied in the following experimental findings: (1) Taurine exhibits significant effects on biochemical systems in vitro. (2) The distribution of taurine is tightly regulated in the different retinal cell types through the development of the retina. (3) Taurine depletion results in significant retinal lesions. (4) Taurine release and uptake has been found to employ distinct regulatory mechanisms in the retina.