P2X 3 receptors desensitize within 100 ms of channel activation, yet recovery from desensitization requires several minutes. The molecular basis for this slow rate of recovery is unknown. We designed experiments to test the hypothesis that this slow recovery is attributable to the high affinity (Ͻ1 nM) of desensitized P2X 3 receptors for agonist. We found that agonist binding to the desensitized state provided a mechanism for potent inhibition of P2X 3 current. Sustained applications of 0.5 nM ATP inhibited Ͼ50% of current to repetitive applications of P2X 3 agonist. Inhibition occurred at 1000-fold lower agonist concentrations than required for channel activation and showed strong use dependence. No inhibition occurred without previous activation and desensitization. Our data are consistent with a model whereby inhibition of P2X 3 by nanomolar [agonist] occurs by the rebinding of agonist to desensitized channels before recovery from desensitization. For several ATP analogs, the concentration required to inhibit P2X 3 current inversely correlated with the rate of recovery from desensitization. This indicates that the affinity of the desensitized state and recovery rate primarily depend on the rate of agonist unbinding. Consistent with this hypothesis, unbinding of [ 32 P]ATP from desensitized P2X 3 receptors mirrored the rate of recovery from desensitization. As expected, disruption of agonist binding by site-directed mutagenesis increased the IC 50 for inhibition and increased the rate of recovery.
Spontaneous electrical activity synchronized among groups of related neurons is a widespread and important feature of central nervous system development. Among the many places from which spontaneous rhythmic activity has been recorded early in development are the cranial motor nerve roots that exit the hindbrain, the motor neuron pool that, at birth, will control the rhythmic motor patterns of swallow, feeding and the oral components of respiratory behaviour. Understanding the mechanism and significance of this hindbrain activity, however, has been hampered by the difficulty of identifying and recording from individual hindbrain motor neurons in living tissue. We have used retrograde labelling to identify living cranial branchiomeric motor neurons in the hindbrain, and [Ca2+]i imaging of such labelled cells to measure spontaneous activity simultaneously in groups of motor neuron somata. We find that branchiomeric motor neurons of the trigeminal and facial nerves generate spontaneous [Ca2+]i transients throughout the developmental period E9.5 to E11.5. During this two‐day period the activity changes from low‐frequency, long‐duration events that are tetrodotoxin insensitive and poorly coordinated among cells, to high‐frequency short‐duration events that are tetrodotoxin sensitive and tightly coordinated thoughout the motor neuron population. This early synchronization may be crucial for correct neuron‐target development.
The -cell ATP-sensitive potassium (K ATP ) channel composed of sulfonylurea receptor SUR1 and potassium channel Kir6.2 serves a key role in insulin secretion regulation by linking glucose metabolism to cell excitability. Mutations in SUR1 or Kir6.2 that decrease channel function are typically associated with congenital hyperinsulinism, whereas those that increase channel function are associated with neonatal diabetes. Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Under normal conditions, both mutant channels showed poor surface expression due to retention in the endoplasmic reticulum, accounting for the loss of channel function phenotype in the congenital hyperinsulinism patients. This trafficking defect, however, could be corrected by treating cells with the oral hypoglycemic drugs sulfonylureas, which we have shown previously to act as small molecule chemical chaperones for K ATP channels. The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6-and 12-fold lower than wild-type channels, respectively. Further analyses revealed a nucleotide-independent decrease in mutant channel intrinsic open probability, suggesting the mutations may reduce ATP sensitivity by causing functional uncoupling between SUR1 and Kir6.2. In insulin-secreting cells, rescue of both mutant channels to the cell surface led to hyperpolarized membrane potentials and reduced insulin secretion upon glucose stimulation. Our results show that sulfonylureas, as chemical chaperones, can dictate manifestation of the two opposite insulin secretion defects by altering the expression levels of the disease mutants.The -cell ATP-sensitive potassium (K ATP ) 3 channels are essential for triggering glucose-stimulated insulin secretion as they couple metabolic signals to electrical signals (1). Each channel is a complex of four regulatory sulfonylurea receptor 1 (SUR1) subunits, encoded by ABCC8, and four pore-forming Kir6.2 inwardly rectifying potassium channel subunits, encoded by KCNJ11 (1). Mutations in ABCC8 or KCNJ11 that perturb the expression and/or gating of the channel lead to channel dysfunction and insulin secretion disorders. Whereas a net loss in channel activity results in congenital hyperinsulinism (CHI), a net gain of channel function causes permanent neonatal diabetes mellitus (PNDM) (2). SUR1 and Kir6.2 co-assemble in the endoplasmic reticulum (ER) to form channel complexes; successful assembly overcomes the arginine-lysine-arginine (RKR) ER retention motif in SUR1 and Kir6.2 to permit channel trafficking to the plasma membrane (3, 4). Quality surveillance mechanisms are in place to prevent misfolded or unassembled channel subunits from exiting the ER where the retained proteins are eventually degraded by the ubiquitin-proteasome pathway (5). In the plasma membrane, channel activities are regulated by i...
The SLC30A8 gene codes for a pancreatic beta-cell-expressed zinc transporter, ZnT8. A polymorphism in the SLC30A8 gene is associated with susceptibility to type 2 diabetes, although the molecular mechanism through which this phenotype is manifest is incompletely understood. Such polymorphisms may exert their effect via impacting expression level of the gene product. We used an shRNA-mediated approach to reproducibly downregulate ZnT8 mRNA expression by >90% in the INS-1 pancreatic beta cell line. The ZnT8-downregulated cells exhibited diminished uptake of exogenous zinc, as determined using the zinc-sensitive reporter dye, zinquin. ZnT8-downregulated cells showed reduced insulin content and decreased insulin secretion (expressed as percent of total insulin content) in response to hyperglycemic stimulus, as determined by insulin immunoassay. ZnT8-depleted cells also showed fewer dense-core vesicles via electron microscopy. These data indicate that reduced ZnT8 expression in cultured pancreatic beta cells gives rise to a reduced insulin response to hyperglycemia. In addition, although we provide no direct evidence, these data suggest that an SLC30A8 expression-level polymorphism could affect insulin secretion and the glycemic response in vivo.
The ATP-sensitive potassium (KATP) channel consisting of the inward rectifier Kir6.2 and SUR1 (sulfonylurea receptor 1) couples cell metabolism to membrane excitability and regulates insulin secretion. Inhibition by intracellular ATP is a hallmark feature of the channel. ATP sensitivity is conferred by Kir6.2 but enhanced by SUR1. The mechanism by which SUR1 increases channel ATP sensitivity is not understood. In this study, we report molecular interactions between SUR1 and Kir6.2 that markedly alter channel ATP sensitivity. Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity ∼100-fold higher than wild-type channels. Cross-linking of E203C in SUR1 and Q52C in Kir6.2 locks the channel in a closed state and is reversible by reducing agents, demonstrating close proximity of the two residues. Our results reveal that ATP sensitivity in KATP channels is a dynamic parameter dictated by interactions between SUR1 and Kir6.2.
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