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. The gene SLC12A6 (solute carrier family 12, member 6), which encodes the K+-Cl- transporter KCC3 and maps within the ACCPN candidate region, was screened for mutations in individuals with ACCPN. Four distinct protein-truncating mutations were found: two in the French Canadian population and two in non-French Canadian families. The functional consequence of the predominant French Canadian mutation (2436delG, Thr813fsX813) was examined by heterologous expression of wildtype and mutant KCC3 in Xenopus laevis oocytes; the truncated mutant is appropriately glycosylated and expressed at the cellular membrane, where it is non-functional. Mice generated with a targeted deletion of Slc12a6 have a locomotor deficit, peripheral neuropathy and a sensorimotor gating deficit, similar to the human disease. Our findings identify mutations in SLC12A6 as the genetic lesion underlying ACCPN and suggest a critical role for SLC12A6 in the development and maintenance of the nervous system.

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 Kir6.2/SUR1 complex. To characterize hit compounds, we developed an assay to quantify endogenous potassium channel activity in INS-1E cells. We quantified ligand-regulated potassium channel activity in INS-1E cells using fluorescence imaging and thallium flux. Potassium channel activity was metabolically regulated and coupled to insulin secretion. The pharmacology of channel opening agents (diazoxide) and closing agents (sulfonylureas) was used to validate the applicability of the assay. A precise high-throughput assay was enabled, and phenotypic screening hits were triaged to enable a higher likelihood of discovering chemical matter with novel and useful mechanisms of action.

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. Here we present the generation of human induced pluripotent stem cell (iPSC) lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterization of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. Cell sorting and gene expression profiling by RNA sequencing revealed that NKX6.1-positive cells from pancreatic differentiations closely resemble human beta cells. Furthermore, functional characterization of the isolated cells demonstrated that glucose-stimulated insulin secretion is mainly confined to the NKX6.1-positive cells. We expect that the NKX6.1-GFP iPSC lines and the results presented here will contribute to the further refinement of differentiation protocols and characterization of hPSC-derived beta cells and motor neurons for disease modelling and cell replacement therapies.

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. We examined the mechanistic basis for the current reduction produced by these variants using the halpha4beta2delta receptor, an isoform more physiologically relevant and linked to epileptogenesis, by characterizing the effects of these variants on receptor cell surface expression and single-channel gating properties. Expression of variant alpha4beta2delta(R220H) receptors resulted in a decrease in surface receptor proteins, and a smaller, but significant, reduction was observed for variant alpha4beta2delta(E177A) receptors. For both variants, no significant alterations of surface expression were observed for mixed population of wild-type and variant receptors. The mean open durations of alpha4beta2delta(E177A) and alpha4beta2delta(R220H) receptor single-channel currents were both significantly decreased compared to wild-type receptors. These data suggest that both delta(E177A) and delta(R220H) variants may result in disinhibition in IGEs by similar cellular and molecular mechanisms, and in heterozygously affected individuals, a reduction in channel open duration of delta subunit-containing GABAA receptors may be the major contributor to the epilepsy phenotypes.

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. Different domains of the gamma2L subunit were replaced by delta subunit sequence, and diazepam sensitivity was determined. Chimeric subunits revealed two areas of interest: domain 1 in transmembrane domain 1 (M1) and domain 2 in the C-terminal portion of transmembrane domain 2 (M2) and the M2-M3 extracellular loop. In those domains, site-directed mutagenesis demonstrated that the following two groups of residues were involved in benzodiazepine transduction of current enhancement: residues Y235, F236, T237 in M1; and S280, T281, I282 in M2 as well as the entire M2-M3 loop. These results suggest that a pocket of residues may transduce benzodiazepine binding to increased gating. Benzodiazepine transduction involves a group of residues that connects the N terminus and M1, and another group of residues that may facilitate an interaction between the N terminus and the M2 and M2-M3 loop domains.

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). Expression of a 1:1 mixture of wild-type andalpha1(A322D) subunits with beta2S and gamma2S subunits (heterozygous transfection) produced smaller currents than wild type and much larger currents than homozygous mutant transfections. Western blot and biotinylation assays demonstrated that the amount of total and surface alpha1 subunit from heterozygous transfections was also intermediate between those of wild-type and homozygous mutant transfections. alpha1(A322D) mutations were then made in covalently tethered triplet (gamma2S-beta2S-alpha1) and tandem (beta2S-alpha1) concatamers to target selectively alpha1(A322D) to each of the asymmetric alpha1 subunits. Coexpression of mutant and wild-type concatamers resulted in expression of either Het(betaalphabeta) or Het(betaalphagamma) receptors. Het(betaalphabeta) currents were smaller than wild type and much larger than Het(betaalphagamma) and homozygous currents. Furthermore, Het(betaalphabeta) transfections contained less beta-alpha concatamer than wild type but more than both Het(betaalphagamma) and homozygous mutant transfections. Thus, whole-cell currents and protein expression of heterozygous alpha1(A322D)beta2Sgamma2S receptors depended on the position of the mutant alpha1 subunit, and GABA(A) receptor currents in heterozygous individuals likely result primarily from wild-type and Het(betaalphabeta) receptors with little contribution from Het(betaalphagamma) and homozygous receptors.

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). Preliminary functional analysis of alpha1beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes suggested that the gamma2 subunit R43Q mutation abolished current enhancement by the benzodiazepine, diazepam, and that the gamma2 subunit K289M mutation decreased current amplitudes. We used single-channel recording and concentration jump techniques applied to outside out patches to evaluate the impact of these mutations on GABA(A) receptor channel function of the highly conserved rat ortholog subunits expressed in human embryonic kidney cells. When coexpressed with alpha1 and beta3 subunits, no differences were observed between wild-type and mutant GABA(A) receptor current activation rates or rates or extent of desensitization during prolonged (400 msec) GABA application (1 mm). Although deactivation after brief (5 msec) or prolonged (400 msec) GABA application was unaltered by the R43Q mutation, deactivation (a correlate of IPSC duration) was accelerated for the K289M mutation. Faster deactivation was likely a consequence of altered gating, because single-channel openings had shorter mean duration. Interestingly, the R43Q mutation did not alter diazepam potentiation. It did, however, substantially decrease current amplitude, which was not caused by decreased single-channel conductance or open time, suggesting reduced surface expression of functional receptors. The two gamma2 subunit mutations likely produce disinhibition and familial epilepsy by distinct mechanisms, suggesting that maintenance of neuronal inhibition depends not only on the peak amplitude of IPSCs, but also on their time course.

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 5.9 kb hKCC2 transcript is specific to brain, and is induced during in vitro differentiation of NT2 teratocarcinoma cells into neuronal NT2-N cells. The 24-exon SLC12A5 gene is on human chromosome 20q13, and contains a polymorphic dinucleotide repeat within intron 1 near a potential binding site for neuron-restrictive silencing factor. Expression of hKCC2 cRNA in Xenopus laevis oocytes results in significant Cl(-)-dependent (86)Rb(+) uptake under isotonic conditions; cell swelling under hypotonic conditions causes a 20-fold activation, which is blocked by the protein phosphatase inhibitor calyculin-A. In contrast, oocytes expressing mouse KCC4 do not mediate isotonic K-Cl cotransport but express much higher absolute transport activity than KCC2 oocytes under hypotonic conditions. Initial and steady state kinetics of hKCC2-injected oocytes were performed in both isotonic and hypotonic conditions, revealing K(m)s for K(+) and Cl(-) of 9.3+/-1.8 mM and 6.8+/-0.9 mM, respectively; both affinities are significantly higher than KCC1 and KCC4. The K(m) for Cl(-) is close to the intracellular Cl(-) activity of mature neurons, as befits a neuronal efflux mechanism.

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. The composite binding surface likely restricts the recruitment of MAML proteins to promoters on which Notch:CSL complexes have been preassembled, ensuring tight transcriptional control of Notch target genes.

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. Total alpha1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 +/- 6%) or when coexpressed with beta2 and gamma2S subunits (78 +/- 6%), indicating an expression reduction prior to subunit oligomerization. In alpha1beta2gamma2S receptors, endoglycosidase H deglycosylated only 26 +/- 5% of alpha1 subunits, consistent with substantial protein maturation, but in alpha1(A322D)beta2gamma2S receptors, endoglycosidase H deglycosylated 91 +/- 4% of alpha1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the alpha1 subunit was tagged with yellow (alpha1-YFP) or cyan (alpha1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 +/- 4% of alpha1-YFPbeta2gamma2 but only 5 +/- 1% alpha1(A322D)-YFPbeta2gamma2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 +/- 4% alpha1-YFPbeta2gamma2S, 86 +/- 3% alpha1(A322D)-YFP). Heterozygous expression of alpha1-CFPbeta2gamma2S and alpha1(A322D)-YFPbeta2gamma2S or alpha1-YFPbeta2gamma2S and alpha1(A322D)-CFPbeta2gamma2S receptors showed that membrane GABA(A) receptors contained primarily wild type alpha1 subunits. These data demonstrate that the A322D mutation reduces alpha1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane alpha1 subunits that are almost exclusively wild type subunits.

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. Northern blot analysis of mouse tissues indicates that KCC3b expression is particularly robust in the kidney, which also expresses KCC3a. Western blotting of mouse tissue using an exon 3-specific antibody reveals that the kidney is also unique in expressing immunoreactive protein of a lower mass, suggestive evidence that the shorter KCC3b protein predominates in kidney. Immunofluorescence reveals basolateral expression of KCC3 protein along the entire length of the proximal tubule, in both the mouse and rat. Removal of the 15-residue exon 2 by alternative splicing generates the KCC3a-x2M and KCC3b-x2M isoforms; other splicing events at an alternative acceptor site within exon 1a generate the KCC3a-S isoform, which is 60 residues shorter than KCC3a. This variation in sequence of NH(2)-terminal cytoplasmic domains occurs proximal to a stretch of highly conserved residues and affects the content of putative phosphorylation sites. Kinetic characterization of KCC3a in X. laevis oocytes reveals apparent K(m)s for Rb(+) and Cl(-) of 10.7 +/- 2.5 and 7.3 +/- 1.2 mM, respectively, with an anion selectivity of Br(-) > Cl(-) > PO(4) = I(-) = SCN(-) = gluconate. All five NH(2)-terminal isoforms are activated by cell swelling (hypotonic conditions), with no activity under isotonic conditions. Although the isoforms do not differ in the osmotic set point of swelling activation, this activation is more rapid for the KCC3a-x2M and KCC3a-S proteins. In summary, there is significant NH(2)-terminal heterogeneity of KCC3, with particularly robust expression of KCC3b in the kidney. Basolateral swelling-activated K(+)-Cl(-) cotransport mediated by KCC3 likely functions in cell volume regulation during the transepithelial transport of both salt and solutes by the proximal tubule.

NSD1 is essential for early post-implantation development and has a catalytically active SET domain.

The nuclear receptor-binding SET domain-containing protein (NSD1) belongs to an emerging family of proteins, which have all been implicated in human malignancy. To gain insight into the biological functions of NSD1, we have generated NSD1-deficient mice by gene disruption. Homozygous mutant NSD1 embryos, which initiate mesoderm formation, display a high incidence of apoptosis and fail to complete gastrulation, indicating that NSD1 is a developmental regulatory protein that exerts function(s) essential for early post-implantation development. We have also examined the enzymatic potential of NSD1 and found that its SET domain possesses intrinsic histone methyltransferase activity with specificity for Lys36 of histone H3 (H3-K36) and Lys20 of histone H4 (H4-K20).

[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, 81.5% (13 cases), 86.7% (13 cases) and 89.5% (17 cases) of the SAA patients reached and maintained normal peripheral blood cell counts, 93.4% (15 cases), 93.3% (14 cases) and 94.7% (18 cases) showed normal bone marrow pictures, and 37.5% (6 cases), 40.0% (6 cases) and 73.7% (14 cases) had normal yields of bone marrow cell culture, respectively. Overall, 86.0% (43 cases), 94.0% (47 cases) and 52.0% (26 cases) of the total SAA patients were normalized in peripheral blood counts, bone marrow picture and culture of hematopoietic progenitor yields, respectively. During the follow-up, 88.0% (44 cases) of the patients achieved 100 of Karnofsky scores; 26 of the 31 patients (83.9%) who received bone marrow biopsy showed normal histological pictures, and 29 of 37 patients (78.4%) tested had normal subsets of T lymphocytes. No clonal disease was found. The late side-effects of IST were mild. All of the parameters tested were normal in 24 patients. CONCLUSION: After IST, the hematopoietic function of bone marrow, the immunity of the T lymphocyte and the life quality were normalized with few side-effects in patients with SAA. These patients would probably be cured.