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.
We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2–M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ∼92-fold increased gating equilibrium constant, which is consistent with an ∼10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening ∼28-fold, slows channel closing ∼3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ∼1.4 × 106 s−1 and close at a rate of ∼760 s−1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ∼140 s−1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2–M3L of the α subunit moves before the corresponding linkers of the β and ε subunits.
Head and neck cancers comprise a complex genetic disease. Although much has been learned about the molecular genetics of head and neck cancers, continued study of multiple genes is critical for further progress. Gene therapy, although promising, must also overcome this complexity.
During routine sequencing of our mouse muscle α subunit acetylcholine receptor channel (AChR) cDNA clones, we detected a discrepancy with the GenBank database entry (accession X03986). At nucleotides 1305‐7 (residue 433, in the M4 domain) the database lists GTC which encodes a valine, while our putative ‘wild‐type’ cDNA had the nucleotides GCC, which encodes an alanine. No other sequence differences were found. PCR amplification of genomic DNA confirmed that the BALB/C mouse α subunit gene has a T nucleotide at position 1306, and, therefore, that the protein has a V at position 433 in the M4 segment. In order to determine the functional consequences of this difference, either wild‐type (V433) or mutant (A433) α subunits were co‐expressed in HEK cells with mouse β, ε and δ subunits. Single‐channel currents were recorded in cell‐attached patches, and rate and equilibrium constants were estimated from open and closed durations obtained from a range of ACh concentrations. No significant differences were found between the activation rate constants or equilibrium constants of the V433 and A433 variants. Kinetic modelling of αV433 AChR suggests that the two transmitter binding sites have similar dissociation equilibrium constants for acetylcholine (∼160 μM, in 142 mM extracellular KCl). Diliganded AChRs occupy a closed state that has a lifetime of ∼1 ms. The rate constants for entering and leaving this state do not vary with the ACh concentration. The kinetics of a mutant AChR that causes a slow channel congenital myaesthenic syndrome, αG153S, was re‐examined. The properties of this mutant were similar with a V or an A at position α433.
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