Tetrodotoxin (TTX)-insensitive slow Na+ channels are converted or replaced by TTX-sensitive fast Na+ channels during normal embryonic development of the chick heart, and rapid reversion occurs in monolayer cell culture (denervated). Fast Na+ channels first appear at 4 to 5 days, which is about the time of innervation. Studies were done to determine whether changes in cation channels will occur while hearts are in organ culture. To test whether fast Na+ channels will develop in the absence of innervation, hearts from chick embryos 2 to 3 days old were placed into culture for 6 to 8 days. Although the resting potentials of the ventricular cells were about the same as those obtained from fresh 8 to 10 day old hearts, the maximum rate of rise of the action potentials () did not reach the high value (about 80 V/s) expected from the calendar age. Instead remained at about the same value (12 V/s) that the hearts had when placed into culture. The action potentials were completely insensitive to TTX. The slow channels admit primarily Na+ and not Ca2+ because Mn2+ (1 mm) and lowering [Ca2+]0 to nearly zero by EGTA did not diminish . To test whether the fast Na+ channels disappear in organ culture, hearts from embryos 15 to 19 days old were cultured as whole hearts or minced hearts. The whole hearts survived well for 1 to 6 days; the values remained high (～ 100 V/s), and TTX completely blocked the action potentials. The minced hearts had variable values, depending on the piece. Those pieces which had a low were insensitive to TTX, and those which had a high or intermediate , were reduced to 5 to 20 V/s by TTX; these persisting responses in TTX were not blocked by Mn2+ or zero [Ca2+]0. The results suggest that, while in organ culture, young hearts do not gain fast Na+ channels or lose the slow Na+ channels that would normally occur in situ. Organ-cultured old hearts left intact do not lose their fast Na+ channels. Thus, young or old hearts retain the channels that they originally possessed when placed into culture. Mincing initiates a gain of slow Na+ channels, and in some pieces, a partial loss of fast Na+ channels. 相似文献
GCK-MODY, dominantly inherited mild hyperglycemia, is associated with more than 600 mutations in the glucokinase gene. Different molecular mechanisms have been shown to explain GCK-MODY. Here, we report a Pakistani family harboring the glucokinase mutation c.823C > T (p.R275C). The recombinant and in cellulo expressed mutant pancreatic enzyme revealed slightly increased enzyme activity (kcat) and normal affinity for α-D-glucose, and resistance to limited proteolysis by trypsin comparable with wild-type. When stably expressed in HEK293 cells and MIN6 β-cells (at different levels), the mutant protein appeared misfolded and unstable with a propensity to form dimers and aggregates. Its degradation rate was increased, involving the lysosomal and proteasomal quality control systems. On mutation, a hydrogen bond between the R275 side-chain and the carbonyl oxygen of D267 is broken, destabilizing the F260-L271 loop structure and the protein. This promotes the formation of dimers/aggregates and suggests that an increased cellular degradation is the molecular mechanism by which R275C causes GCK-MODY. 相似文献
