Nicotine obtained from tobacco can improve learning and memory on various tasks and has been linked to arousal, attention, rapid information processing, working memory, and long-term memories that can cause craving years after someone has stopped smoking. One likely target for these effects is the hippocampus, a centre for learning and memory that has rich cholinergic innervation and dense nicotinic acetylcholine receptor (nAChR) expression. During Alzheimer's dementia there are fewer nAChRs and the cholinergic inputs to the hippocampus degenerate. However, there is no evidence for fast synaptic transmission mediated by nAChRs in the hippocampus, and their role is not understood. Nicotine is known to act on presynaptic nAChRs within the habenula of chick to enhance glutamatergic transmission; here we report that a similar mechanism operates in the hippocampus. Measurements of intracellular Ca2+ in single mossy-fibre presynaptic terminals indicate that nAChRs containing the alpha7 subunit can mediate a Ca2+ influx that is sufficient to induce vesicular neurotransmitter release. We propose that nicotine from tobacco influences cognition by enhancing synaptic transmission. Conversely, a decreased efficacy of transmission may account for the deficits associated with the loss of cholinergic innervation during Alzheimer's disease.

Grayanotoxin I (GTX I) is a diterpenoid extracted from the family of Ericaceae that binds to Na + channels and causes persistent activation. We investigated the interaction of GTX I with the amino acid residues I1575, F1579 and Y1586 in transmembrane segment D4S6 of W W1. In F1579A, GTX shifted the threshold potential about 50 mV in the hyperpolarizing direction and modified Na + channels twice as efficiently as that in wild-type. In contrast, these GTX-effects were eliminated completely in the I1575A mutant and were reduced substantially in mutant Y1586A. Lysine substitution for F1579 significantly reduced and for Y1586 completely eradicated the GTX-effect. Our data suggest that the GTX receptor site shares overlapping but non-identical molecular determinants with BTX in D4S6 and has common molecular determinants in D1S6.z 2000 Federation of European Biochemical Societies.

The effect of grayanotoxin (GTX) on site-specific mutants of the alpha-subunit of rat skeletal muscle Na(+) channels (micro1) (micro1-I433K, micro1-N434K and micro1-L437K), which are resistant to batrachotoxin (BTX) (Wang and Wang (1998) Proc Natl Acad Sci USA, 95, 2653-2658) was studied using a whole-cell patch-clamp method. The GTX modification of the Na(+) channels was detected as a characteristic-sustained Na(+) current flow with repetitive pulses. We also studied the GTX action on mutants of the alpha-subunit of rat heart Na(+) channels (RH1) (RH1-V406K and RH1-L410K) which match with micro1-I433 and micro1-L437. All the mutants lost their sensitivity to GTX. This finding indicates that GTX may share a binding site with BTX in transmembrane segment I-S6 of two different Na(+) channel isoforms, micro1 and RH1.

The effects of sulfhydryl (SH) reagents on the L-type Ca current (ICa) were studied in frog ventricular myocytes using the whole-cell patch-clamp method. Methanethiosulfonate ethylammonium (MTSEA+) was found to enter the cell through the membrane and cause a remarkable increase in Ica from the intracellular side. Methanethiosulfonate ethyltrimethylammonium (MTSET+) and methanethiosulfonate ethylsulfonate (MTSES-) could not penetrate the membrane and were effective only when directly applied to the intracellular side. In addition, suppressive effects on ICa of these MTS reagents were indicated by the following observation. A progressive decay in the peak amplitude of ICa after establishing maximal ICa, stimulated by intracellular MTSET+, was prevented by adding extracellular dithiothreitol (DTT). The SH-oxidizing agents N-ethylmaleimide (NEM), chloramine-T (CL-T), 2,2'-dithiodipyridine (DTDP) and 2,2'-dithio-bis-5-nitropyridine (DTBNP) also exerted a stimulatory effect on Ica. The effect of SH reagents persisted even when cAMP production was inhibited with Rp-cAMP-S, or when G-protein was inhibited with 1 mM GDPbetaS, indicating that the effect is not due to cAMP production or G-protein stimulation. It is concluded that there are sites on the Ca channels that are subject to direct modification by SH reagents.

Distinct properties of grayanotoxin (GTX) among other lipid‐soluble toxins were elucidated by quantitative analysis made on the Na+ channel in frog ventricular myocytes. GTX‐modified current (IGTX) was induced strictly in proportion to the open probability of Na+ channels during preconditioning pulses irrespective of its duration, amplitude or partial removal of inactivation by chloramine‐T. This confirms that GTX binds to the Na+ channel exclusively in its open state, while batrachotoxin (BTX) was reported to be capable of modifying slow‐inactivated Na+ channels, and veratridine exhibited voltage‐dependent modification. The GTX‐modified channel did not show any inactivation property, which is different from reported results with veratridine and BTX. Estimated unbinding rates of GTX were in reverse proportion to the activation curve of GTX‐modified Na+ channels. This was not the previously reported case with veratridine. A model including unbinding kinetics of GTX and slow inactivation of unmodified Na+ channels in which GTX was permitted to bind only to the open state of Na+ channels indicated that unbinding reactions of GTX occur only in the closed state.