A High-Throughput Assay for the Pancreatic Islet Beta-Cell Potassium Channel: Use in the Pharmacological Characterization of Insulin Secretagogues Identified from Phenotypic Screening.

Phenotypic screening is a neoclassical approach for drug discovery. We conducted phenotypic screening for insulin secretion enhancing agents using INS-1E insulinoma cells as a model system for pancreatic beta-cells. A principal regulator of insulin secretion in beta-cells is the metabolically regulated potassium channel Kir6.

NKX6.1 induced pluripotent stem cell reporter lines for isolation and analysis of functionally relevant neuronal and pancreas populations.

Recent studies have reported significant advances in the differentiation of human pluripotent stem cells to clinically relevant cell types such as the insulin producing beta-like cells and motor neurons. However, many of the current differentiation protocols lead to heterogeneous cell cultures containing cell types other than the targeted cell fate. Genetically modified human pluripotent stem cells reporting the expression of specific genes are of great value for differentiation protocol optimization and for the purification of relevant cell populations from heterogeneous cell cultures.

Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes.

Notch receptors transduce essential developmental signals between neighboring cells by forming a complex that leads to transcription of target genes upon activation. We report here the crystal structure of a Notch transcriptional activation complex containing the ankyrin domain of human Notch1 (ANK), the transcription factor CSL on cognate DNA, and a polypeptide from the coactivator Mastermind-like-1 (MAML-1). Together, CSL and ANK create a groove to bind the MAML-1 polypeptide as a kinked, 70 A helix.

Delta subunit susceptibility variants E177A and R220H associated with complex epilepsy alter channel gating and surface expression of alpha4beta2delta GABAA receptors.

Most human idiopathic generalized epilepsies (IGEs) are polygenic, but virtually nothing is known of the molecular basis for any of the complex epilepsies. Recently, two GABAA receptor delta subunit variants (E177A, R220H) were proposed as susceptibility alleles for generalized epilepsy with febrile seizures plus and juvenile myoclonic epilepsy. In human embryonic kidney 293T cells, recombinant halpha1beta2delta(E177A) and halpha1beta2delta(R220H) receptor currents were reduced, but the basis for the current reduction was not determined.

Structural determinants of benzodiazepine allosteric regulation of GABA(A) receptor currents.

Benzodiazepine enhancement of GABA(A) receptor current requires a gamma subunit, and replacement of the gamma subunit by the delta subunit abolishes benzodiazepine enhancement. Although it has been demonstrated that benzodiazepines bind to GABA(A) receptors at the junction between alpha and gamma subunits, the structural basis for the coupling of benzodiazepine binding to allosteric enhancement of the GABA(A) receptor current is unclear. To determine the structural basis for this coupling, the present study used a chimera strategy, using gamma2L-delta GABA(A) receptor subunit chimeras coexpressed with alpha1 and beta3 subunits in human embryonic kidney 293T cells.

Endoplasmic reticulum retention and associated degradation of a GABAA receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the alpha1 subunit.

A GABA(A) receptor alpha1 subunit epilepsy mutation (alpha1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the alpha1 subunit. We reported previously that heterologous expression of alpha1(A322D)beta2gamma2 receptors in mammalian cells resulted in reduced total and surface alpha1 subunit protein. Here we demonstrate the mechanism of this reduction.

NH2-terminal heterogeneity in the KCC3 K+-Cl- cotransporter.

The SLC12A6 gene encoding the K(+)-Cl(-) cotransporter KCC3 is expressed in multiple tissues, including kidney. Here, we report the molecular characterization of several NH(2)-terminal isoforms of human and mouse KCC3, along with intrarenal localization and functional characterization in Xenopus laevis oocytes. Two major isoforms, KCC3a and KCC3b, are generated by transcriptional initiation 5' of two distinct first coding exons.

The juvenile myoclonic epilepsy GABA(A) receptor alpha1 subunit mutation A322D produces asymmetrical, subunit position-dependent reduction of heterozygous receptor currents and alpha1 subunit protein expression.

Individuals with autosomal dominant juvenile myoclonic epilepsy are heterozygous for a GABA(A) receptor alpha1 subunit mutation (alpha1A322D). GABA(A) receptor alphabetagamma subunits are arranged around the pore in a beta-alpha-beta-alpha-gamma sequence (counterclockwise from the synaptic cleft). Therefore, each alpha1 subunit has different adjacent subunits, and heterozygous expression of alpha1(A322D), beta, and gamma subunits could produce receptors with four different subunit arrangements: beta-alpha1-beta-alpha1-gamma (wild type); beta-alpha1(A322D)-beta-alpha1-gamma (Het(betaalphabeta)); beta-alpha1-beta-alpha1(A322D)-gamma (Het(betaalphagamma));beta-alpha1(A322D)-beta-alpha1(A322D)-gamma (homozygous).

The K-Cl cotransporter KCC3 is mutant in a severe peripheral neuropathy associated with agenesis of the corpus callosum.

Peripheral neuropathy associated with agenesis of the corpus callosum (ACCPN) is a severe sensorimotor neuropathy associated with mental retardation, dysmorphic features and complete or partial agenesis of the corpus callosum. ACCPN is transmitted in an autosomal recessive fashion and is found at a high frequency in the province of Quebec, Canada. ACCPN has been previously mapped to chromosome 15q.

[A long-term follow-up study of 50 patients with severe aplastic anemia who have survived more than 3 years after immunosuppressive therapy].

Objective: To evaluate the long-term outcome of immunosuppressive therapy (IST) in patients with severe aplastic anemia (SAA).

Methods: Hematopoietic recovery (peripheral blood cell counts, bone marrow aspirates, bone marrow biopsy, in vitro culture of hematopoietic progenitors), immunity of T lymphocyte, quality of life and side-effects of the therapy were assessed in 50 SAA patients who have survived more than 3 years after IST.

Results: At 3 years, 4 years and 5 years follow-up, 81.

Molecular, functional, and genomic characterization of human KCC2, the neuronal K-Cl cotransporter.

The expression level of the neuronal-specific K-Cl cotransporter KCC2 (SLC12A5) is a major determinant of whether neurons will respond to GABA with a depolarizing, excitatory response or a hyperpolarizing, inhibitory response. In view of the potential role in human neuronal excitability we have characterized the hKCC2 cDNA and gene. The 5.

Two different mechanisms of disinhibition produced by GABAA receptor mutations linked to epilepsy in humans.

The first mutations of the GABA(A) receptor channel linked to familial epilepsy in humans were reported recently (Baulac et al., 2001; Wallace et al., 2001).