Structurally distinct ligands rescue biogenesis defects of the KATP channel complex via a converging mechanism.

Small molecules that correct protein misfolding and misprocessing defects offer a potential therapy for numerous human diseases. However, mechanisms underlying pharmacological correction of such defects, especially in heteromeric complexes with structurally diverse constituent proteins, are not well understood. Here we investigate how two chemically distinct compounds, glibenclamide and carbamazepine, correct biogenesis defects in ATP-sensitive potassium (KATP) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2. We present evidence that despite structural differences, carbamazepine and glibenclamide compete for binding to KATP channels, and both drugs share a binding pocket in SUR1 to exert their effects. Moreover, both compounds engage Kir6.2, in particular the distal N terminus of Kir6.2, which is involved in normal channel biogenesis, for their chaperoning effects on SUR1 mutants. Conversely, both drugs can correct channel biogenesis defects caused by Kir6.2 mutations in a SUR1-dependent manner. Using an unnatural, photocross-linkable amino acid, azidophenylalanine, genetically encoded in Kir6.2, we demonstrate in living cells that both drugs promote interactions between the distal N terminus of Kir6.2 and SUR1. These findings reveal a converging pharmacological chaperoning mechanism wherein glibenclamide and carbamazepine stabilize the heteromeric subunit interface critical for channel biogenesis to overcome defective biogenesis caused by mutations in individual subunits.

Carbamazepine inhibits ATP-sensitive potassium channel activity by disrupting channel response to MgADP.

In pancreatic ß-cells, K(ATP) channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. Sulfonylureas, insulin secretagogues used to treat type II diabetes, inhibit K(ATP) channel activity primarily by abolishing the stimulatory effect of MgADP endowed by SUR1. In addition, sulfonylureas have been shown to function as pharmacological chaperones to correct channel biogenesis and trafficking defects. Recently, we reported that carbamazepine, an anticonvulsant known to inhibit voltage-gated sodium channels, has profound effects on K(ATP) channels. Like sulfonylureas, carbamazepine corrects trafficking defects in channels bearing mutations in the first transmembrane domain of SUR1. Moreover, carbamazepine inhibits the activity of K(ATP) channels such that rescued mutant channels are unable to open when the intracellular ATP/ADP ratio is lowered by metabolic inhibition. Here, we investigated the mechanism by which carbamazepine inhibits K(ATP) channel activity. We show that carbamazepine specifically blocks channel response to MgADP. This gating effect resembles that of sulfonylureas. Our results reveal striking similarities between carbamazepine and sulfonylureas in their effects on K(ATP) channel biogenesis and gating and suggest that the 2 classes of drugs may act via a converging mechanism.

Carbamazepine as a novel small molecule corrector of trafficking-impaired ATP-sensitive potassium channels identified in congenital hyperinsulinism.

ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to ß-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human ß-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients.

Role of Derlin-1 protein in proteostasis regulation of ATP-sensitive potassium channels.

ATP-sensitive potassium (K(ATP)) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2 regulate insulin secretion by linking glucose metabolism with membrane potential. The number of K(ATP) channels in the plasma membrane affects the sensitivity of ß-cells to glucose. Aberrant surface channel expression leads to insulin secretion disease. Previously, we have shown that K(ATP) channel proteins undergo endoplasmic reticulum (ER)-associated degradation (ERAD) via the ubiquitin-proteasome pathway, and inhibition of proteasome function results in an increase in channel surface expression. Here, we investigated whether Derlin-1, a protein involved in retrotranslocation of misfolded or misassembled proteins across the ER membrane for degradation by cytosolic proteasomes, plays a role in ERAD and, in turn, biogenesis efficiency of K(ATP) channels. We show that both SUR1 and Kir6.2 form a complex with Derlin-1 and an associated AAA-ATPase, p97. Overexpression of Derlin-1 led to a decrease in the biogenesis efficiency and surface expression of K(ATP) channels. Conversely, knockdown of Derlin-1 by RNA interference resulted in increased processing of SUR1 and a corresponding increase in surface expression of K(ATP) channels. Importantly, knockdown of Derlin-1 increased the abundance of disease-causing misfolded SUR1 or Kir6.2 proteins and even partially rescued surface expression in a mutant channel. We conclude that Derlin-1, by being involved in ERAD of SUR1 and Kir6.2, has a role in modulating the biogenesis efficiency and surface expression of K(ATP) channels. The results suggest that physiological or pathological changes in Derlin-1 expression levels may affect glucose-stimulated insulin secretion by altering surface expression of K(ATP) channels.

Nonintegrating foamy virus vectors.

Foamy viruses (FVs), or spumaviruses, are integrating retroviruses that have been developed as vectors. Here we generated nonintegrating foamy virus (NIFV) vectors by introducing point mutations into the highly conserved DD35E catalytic core motif of the foamy virus integrase sequence. NIFV vectors produced high-titer stocks, transduced dividing cells, and did not integrate. Cells infected with NIFV vectors contained episomal vector genomes that consisted of linear, 1-long-terminal-repeat (1-LTR), and 2-LTR circular DNAs. These episomes expressed transgenes, were stable, and became progressively diluted in the dividing cell population. 1-LTR circles but not 2-LTR circles were found in all vector stocks prior to infection. Residual integration of NIFV vectors occurred at a frequency 4 logs lower than that of integrase-proficient FV vectors. Cre recombinase expressed from a NIFV vector mediated excision of both an integrated, floxed FV vector and a gene-targeted neo expression cassette, demonstrating the utility of these episomal vectors. The broad host range and large packaging capacity of NIFV vectors should make them useful for a variety of applications requiring transient gene expression.

Successful treatment of canine leukocyte adhesion deficiency by foamy virus vectors.

Recent successes in treating genetic immunodeficiencies have demonstrated the therapeutic potential of stem cell gene therapy. However, the use of gammaretroviral vectors in these trials led to insertional activation of nearby oncogenes and leukemias in some study subjects, prompting studies of modified or alternative vector systems. Here we describe the use of foamy virus vectors to treat canine leukocyte adhesion deficiency (CLAD). Four of five dogs with CLAD that received nonmyeloablative conditioning and infusion of autologous, CD34+ hematopoietic stem cells transduced by a foamy virus vector expressing canine CD18 had complete reversal of the CLAD phenotype, which was sustained more than 2 years after infusion. In vitro assays showed correction of the lymphocyte proliferation and neutrophil adhesion defects that characterize CLAD. There were no genotoxic complications, and integration site analysis showed polyclonality of transduced cells and a decreased risk of integration near oncogenes as compared to gammaretroviral vectors. These results represent the first successful use of a foamy virus vector to treat a genetic disease, to our knowledge, and suggest that foamy virus vectors will be effective in treating human hematopoietic diseases.

Reduction of radiculopathy and pain with Oxiplex/SP gel after laminectomy, laminotomy, and discectomy: a pilot clinical study.

STUDY DESIGN: Safety using Oxiplex/SP Gel during single-level discectomy for reduction of symptoms associated with unilateral herniation of the lumbar disc was investigated by self-assessment questionnaire and magnetic resonance imaging. OBJECTIVE: To evaluate the safety and assess the efficacy parameters of Oxiplex/SP Gel. SUMMARY OF BACKGROUND DATA: Animal studies demonstrated that Oxiplex/SP Gel (CMC/PEO) reduced epidural fibrosis after lumbar surgery. METHODS: Surgeons examined spine and lower extremities of patients scheduled for discectomy to assess neurologic function and pain. Treated patients received sufficient Oxiplex/SP Gel (1-3 mL) to coat the nerve root and fill the epidural space. The control condition was surgery alone. At baseline, then 30 days, 90 days, and 6 months after surgery, patients completed self-assessment questionnaires concerning leg pain, lower extremity weakness, functional disability, daily living activities, symptoms, and radiculopathy. Magnetic resonance imaging was performed at baseline and 90 days after surgery. At 30 and 90 days after surgery, patients underwent physical examination, wound inspection, and laboratory tests. RESULTS: The surgical procedures were well tolerated by the 23 patients treated with Oxiplex/SP Gel and the 11 control patients. There were no unanticipated adverse events, no clinically significant laboratory results, and no significant differences detected by magnetic resonance imaging. Treated patients had greater reduction in outcome measures at 30 days. The differences in scores were attenuated at 90 days and 6 months. A subgroup, the patients with significant leg pain and weakness at baseline (11 patients treated with Oxiplex/SP Gel and 7 control patients), had greater reduction in outcome measures than the control patients throughout the study. CONCLUSIONS: Oxiplex/SP Gel was easy to use and safe for patients undergoing unilateral discectomy. Greater benefit in clinical outcome measures was seen in gel-treated patients, especially those with severe leg pain and weakness at baseline.