ClC-1 mutations in myotonia congenita patients: insights into molecular gating mechanisms and genotype-phenotype correlation.

KEY POINTS: Loss-of-function mutations of the skeletal muscle ClC-1 channel cause myotonia congenita with variable phenotypes. Using patch clamp we show that F484L, located in the conducting pore, probably induces mild dominant myotonia by right-shifting the slow gating of ClC-1 channel, without exerting a dominant-negative effect on the wild-type (WT) subunit. Molecular dynamics simulations suggest that F484L affects the slow gate by increasing the frequency and the stability of H-bond formation between E232 in helix F and Y578 in helix R. Three other myotonic ClC-1 mutations are shown to produce distinct effects on channel function: L198P shifts the slow gate to positive potentials, V640G reduces channel activity, while L628P displays a WT-like behaviour (electrophysiology data only). Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function. ABSTRACT: Myotonia congenita is an inherited disease caused by loss-of-function mutations of the skeletal muscle ClC-1 chloride channel, characterized by impaired muscle relaxation after contraction and stiffness. In the present study, we provided an in-depth characterization of F484L, a mutation previously identified in dominant myotonia, in order to define the genotype-phenotype correlation, and to elucidate the contribution of this pore residue to the mechanisms of ClC-1 gating. Patch-clamp recordings showed that F484L reduced chloride currents at every tested potential and dramatically right-shifted the voltage dependence of slow gating, thus contributing to the mild clinical phenotype of affected heterozygote carriers. Unlike dominant mutations located at the dimer interface, no dominant-negative effect was observed when F484L mutant subunits were co-expressed with wild type. Molecular dynamics simulations further revealed that F484L affected the slow gate by increasing the frequency and stability of the H-bond formation between the pore residue E232 and the R helix residue Y578. In addition, using patch-clamp electrophysiology, we characterized three other myotonic ClC-1 mutations. We proved that the dominant L198P mutation in the channel pore also right-shifted the voltage dependence of slow gating, recapitulating mild myotonia. The recessive V640G mutant drastically reduced channel function, which probably accounts for myotonia. In contrast, the recessive L628P mutant produced currents very similar to wild type, suggesting that the occurrence of the compound truncating mutation (Q812X) or other muscle-specific mechanisms accounted for the severe symptoms observed in this family. Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function.

Use of a standardized protocol to decrease medication errors and adverse events related to sliding scale insulin.

PROBLEM: Sliding scale insulin (SSI) is frequently used for inpatient management of hyperglycemia and is associated with a large number of medication errors and adverse events including hypoglycemia and hyperglycemia. DESIGN: Observational before and after study evaluating the impact of implementation of a standardized SSI protocol and preprinted physician order form. SETTING: University Hospital in Pittsburgh, PA, USA. STRATEGY FOR CHANGE: Guidelines for the use of SSI were created by an interdisciplinary committee and implemented in non-intensive care units. In addition, a preprinted physician order sheet was developed which included the guidelines and an option for ordering one of three standardized insulin sliding scales or a patient specific scale. EFFECT OF CHANGE: One year after implementation the physician order form was used for 91% of orders and, overall, 86% of SSI orders followed the guidelines. The number of prescribing errors found on chart review was reduced from 10.3 per 100 SSI patient-days at baseline to 1.2 at 1 year (p = 0.03). The number of hyperglycemia episodes 1 year after implementation decreased from 55.9 to 16.3 per 100 SSI patient-days. LESSONS LEARNT: The protocol was readily accepted by hospital staff and was associated with decreased prescribing errors and decreased frequency of hyperglycemia.

In vivo silencing of aquaporin-1 by RNA interference inhibits angiogenesis in the chick embryo chorioallantoic membrane assay.

Aquaporin-1 (AQP1) is a water channel protein mainly expressed in endothelial and epithelial cells of many tissues, including the vasculature where it serves to increase cell membrane water permeability. Previous studies in active multiple myeloma patients and in AQP1 KO mice indicated an involvement of AQP1 in physiological and tumor angiogenesis. To understand the physiological role of AQP1 in angiogenesis, we used a 21-nucleotide small interfering RNA duplexes (siRNA) to knockdown AQP1 in the chick embryo chorioallantoic membrane (CAM), a commonly used in vivo assay to study both angiogenic and angiostatic molecules. Chicken AQP1 sequence was identified and utilized to synthesize a siRNA directed to the AQP1 sequence. We then tested the efficiency of the siRNA in vitro, using an AQP1 transfected cell line. The level of AQP1 protein reduction obtained using siRNA was 98 % and 92 % after 1 and 2 day transfection respectively. RNA interference experiments were then performed in vivo by using the CAM assay. Results showed that after 4 days of treatment, AQP1 siRNA was able to strongly inhibit angiogenesis. This is the first study showing the in vivo use of RNA interference technique in the CAM assay. Our results strongly support the hypothesis that AQP1 could have a key role in physiological and pathological angiogenesis.

Study of the mitochondrial transcription factor A (Tfam) gene in the primate Presbytis cristata.

The mitochondrial transcription factor A (Tfam) is a member of the HMG-box protein family, necessary for both transcription and maintenance of mitochondrial DNA. The gene is structured in seven exons and six introns and it is estimated to span about 10 kb in mouse, human and rat. In addition to the full length mRNA of Tfam, a shorter mRNA isoform lacking exon 5 has been found to be widely distributed in human and rat tissues. Here we present the isolation and characterization of Tfam gene in the primate Presbytis cristata which belongs to the Cercopithecidae family. We have determined the complete CDS sequence, the size of all the six introns, the complete sequences of the three shorter ones (I, III, VI) and the partial sequences of the long introns (II, IV, V). The comparison with other available Tfam sequences from mammals has revealed a high degree of conservation (above 90%) both in CDS and introns. By in situ hybridization (FISH) experiments we have mapped Tfam gene on chromosome 12 which, according to other cytogenetics studies, is the homologous region of chromosome 10, where human Tfam has been mapped. Moreover we have searched for the presence of alternatively spliced isoforms through several approaches, such as RT-PCR and differential hybridization. In Presbytis cristata we have not detected the presence of any spliced isoforms lacking exons; however we have identified one isoform in which part of the intron I is retained in the mRNA. The inclusion of this portion of intron I would originate an early stop codon if translated.