A re‐examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics

Abstract

1. Cell-attached single-channel recordings of NMDA channels were carried out in human dentate gyrus granule cells acutely dissociated from slices prepared from hippocampi surgically removed for the treatment of temporal lobe epilepsy (TLE). The channels were activated by ¬_aspartate (250-500 nÒ) in the presence of saturating glycine (8 ìÒ). 2. The main conductance was 51 ± 3 pS. In ten of thirty granule cells, clear subconductance states were observed with a mean conductance of 42 ± 3 pS, representing 8 ± 2 % of the total openings. 3. The mean open times varied from cell to cell, possibly owing to differences in the epileptogenicity of the tissue of origin. The mean open time was 2·70 ± 0·95 ms (range, 1·24-4·78 ms). In 87% of the cells, three exponential components were required to fit the apparent open time distributions. In the remaining neurons, as in control rat granule cells, two exponentials were sufficient. Shut time distributions were fitted by five exponential components. 4. The average numbers of openings in bursts (1·74 ± 0·09) and clusters (3·06 ± 0·26) were similar to values obtained in rodents. The mean burst (6·66 ± 0·9 ms), cluster (20·1 ± 3·3 ms) and supercluster lengths (116·7 ± 17·5 ms) were longer than those in control rat granule cells, but approached the values previously reported for TLE (kindled) rats. 5. As in rat NMDA channels, adjacent open and shut intervals appeared to be inversely related to each other, but it was only the relative areas of the three open time constants that changed with adjacent shut time intervals. 6. The long openings of human TLE NMDA channels resembled those produced by calcineurin inhibitors in control rat granule cells. Yet the calcineurin inhibitor FK_506 (500 nÒ) did not prolong the openings of human channels, consistent with a decreased calcineurin activity in human TLE. 7. Many properties of the human NMDA channels resemble those recorded in rat hippocampal neurons. Both have similar slope conductances, five exponential shut time distributions, complex groupings of openings, and a comparable number of openings per grouping. Other properties of human TLE NMDA channels correspond to those observed in kindling; the openings are considerably long, requiring an additional exponential component to fit their distributions, and inhibition of calcineurin is without effect in prolonging the openings.

Aromaticity at the Water-Hydrocarbon Core Interface of the Membrane: Consequences on the Nicotinic Acetylcholine Receptor

“…The blocking and binding constants were slightly affected, consistent with other studies demonstrating that mutations at the αM4 domain do not modify significantly the binding sites or the ion pore. 22,34,40,41 The same effects on the effective opening and closing rate constants produced by αF426W were also observed in another study using choline as the agonist. 25 On the basis of our results and the aforementioned studies, we demonstrated that position αF426 is highly vulnerable to changes in side-chain identity, and that the increase in receptor functionality arises from an optimal channel transition from the closed to the open state; however, allosteric transitions within these domains are also possible and should not be excluded.…”

Nicotinic Acetylcholine Receptors

“…Salamone et al, 1999;Hatton et al 2003). This is not much different from the value originally found in frog muscle (30000 s −1 at 11° C; Colquhoun & Sakmann, 1985).…”

“…Colquhoun & Sakmann (1985) found little evidence for a difference in frog muscle receptors. Using more recent methods, it has been suggested that there is little difference between the two sites in the adult form of the mouse receptor (Salamone et al, 1999). These authors found a significantly better fit if the sites were not assumed to be equivalent, but had similar equilibrium constants for binding.…”

“…The scheme shown in Fig 10B seems to be adequate to describe the behaviour of the wild type human receptor, though the results of Hatton et al (2003) suggest that the entire concentration range can be fitted well only if either (a) the rate constants for binding to site a depend on whether site b is occupied, or (b) an extra shut state with a lifetime of around 1 ms is added to the right of the open state (Salamone et al, 1999).…”

“…It leads to prolonged decay of miniature end-plate currents (MEPCs) as a result of the channel activations (bursts) being about 15-fold longer than in wild type, on average (Sine et al, 1995b;Croxen et al, 1997). Single channel analysis indicates that the main reason for the prolonged bursts of openings, in receptors that contain αG153S, is slowed dissociation of ACh from the doublyliganded shut state, so the channel re-opens more often (Sine et al, 1995b;Salamone et al, 1999). The fits in the latter paper suggest a 30-fold decrease in equilibrium dissociation constant for ACh binding, attributable mainly to a reduced dissociation rate from the doubly-liganded shut state, with relatively little effect on gating (the opening rate β 2 is hardly changed by the mutation, but openings become somewhat shorter, resulting in about 3-fold increase in E 2 ).…”