Modulation of the gating of CIC-1 by S-(-) 2-(4-chlorophenoxy) propionic acid.

1. Using whole-cell patch-clamping and Sf-9 cells expressing the rat skeletal muscle chloride channel, rCIC-1, the cellular mechanism responsible for the myotonic side effects of clofibrate derivatives was examined. 2. RS-(+/-) 2-(4-chlorophenoxy)propionic acid (RS-(+/-) CPP) and its S-(-) enantiomer produced pronounced effects on CIC-1 gating. Both compounds caused the channels to deactivate more rapidly at hyperpolarizing potentials, which showed as a decrease in the time constants of both the fast and slow deactivating components of the whole cell currents. Both compounds also produced a concentration-dependent shift in the voltage dependence of channel apparent open probability to more depolarizing potentials, with an EC50 of 0.79 and 0.21 mM for the racemate and S-(-) enantiomer respectively. R-(+) CPP at similar concentrations had no effect on gating. RS-(+/-) CPP did not block the passage of Cl- through the pore of rCIC-1. 3. CIC-1 is gated by Cl- binding to a site within an access channel and S-(-) CPP alters gating of the channel by decreasing the affinity of this binding site for Cl-. Comparison of the EC50 for RS-(+/-) CPP and S-(-) CPP indicates that R-(+) CPP can compete with the S-(-) enantiomer for the site but that it is without biological activity. 4. RS-(+/-) CPP produced the same effect on rCIC-1 gating when added to the interior of the cell and in the extracellular solution. 5. S-(-) CPP modulates the gating of CIC-1 to decrease the membrane Cl- conductance (GCl), which would account for the myotonic side effects of clofibrate and its derivatives.

pH-dependent interactions of Cd2+ and a carboxylate blocker with the rat C1C-1 chloride channel and its R304E mutant in the Sf-9 insect cell line.

1. Gating of the skeletal muscle chloride channel (ClC-1) is sensitive to extracellular pH. In this study, whole-cell recording of currents from wild-type (WT) ClC-1 and a mutant, R304E, expressed in the Sf-9 insect cell line was used to investigate further the nature of the pH-sensitive residues. 2. Extracellular Cd2+ produced a concentration-dependent block of WT ClC-1 with an IC50 of 1.0 +/- 0.1 mM and a Hill coefficient of 2.0 +/- 0.3. This block was sensitive to external pH, reducing at low pH, with an apparent pKa of 6.8 +/- 0.1 and a Hill coefficient for proton binding of 3.0 +/- 0.3. Anthracene-9-carboxylate (A-9-C) block of WT ClC-1 was also pH sensitive, increasing at low pH, with an apparent pKa of 6.4 +/- 0.1 and a Hill coefficient for proton binding of 1.0 +/- 0.2. 3. Compared with WT ClC-1, R304E had a lower affinity for Cd2+ (IC50, 3.0 +/- 0.3 mM) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd2+ binding site was reduced to 1.4 +/- 0.3. In contrast, the A-9-C binding site in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WT ClC-1. 4. ClC-1 has at least two binding sites for Cd2+, each of which has at least three residues which can be protonated. Binding of A-9-C is influenced by protonation of a single residue. Arg 304 is not sufficiently close to the A-9-C binding site to affect its characteristics, but it does. alter Cd2+ binding, indicating that transition metal ions and aromatic carboxylates interact with distinct sites. 5. The block of ClC-1 by transition metal ions and the apparent pKa of this block, together with the apparent pKa for A-9-C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC-1.

Concentration and pH dependence of skeletal muscle chloride channel ClC-1.

1. The influence of Cl- concentration and pH on gating of the skeletal muscle Cl- channel, ClC-1, has been assessed using the voltage-clamp technique and the Sf-9 insect cell and Xenopus oocyte expression systems. 2. Hyperpolarization induces deactivating inward currents comprising a steady-state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strongly voltage dependent. 3. Open probability (Po) and kinetics depend on external but not internal Cl- concentration. 4. A point mutation, K585E, in human ClC-1, equivalent to a previously described mutation in the Torpedo electroplaque chloride channel, ClC-0, alters the I-V relationship and kinetics, but retains external Cl- dependence. 5. When external pH is reduced, the deactivating inward currents of ClC-1 are diminished without change in time constants while the steady-state component is enhanced. 6. In contrast, reduced internal pH slows deactivating current kinetics as its most immediately obvious action and the Po curve is shifted in the hyperpolarizing direction. Addition of internal benzoate at low internal pH counteracts both these effects. 7. A current activated by hyperpolarization can be revealed at an external pH of 5.5 in ClC-1, which in some ways resembles currents due to the slow gates of ClC-0. 8. Gating appears to be controlled by a Cl(-)-binding site accessible only from the exterior and, possibly, by modification of this site by external protonation. Intracellular hydroxyl ions strongly affect gating either allosterically or by direct binding and blocking of the pore, an action mimicked by intracellular benzoate.

Characteristics of skeletal muscle chloride channel C1C-1 and point mutant R304E expressed in Sf-9 insect cells.

Using the baculovirus system, the skeletal muscle chloride channel, CIC-1 (rat), and a point mutant replacing arginine 304 with glutamic acid were expressed at high levels in cultured Sf-9 insect cells. Whole-cell patch-clamping revealed large inwardly rectifying currents with maxima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produced inactivating currents made up of a steady state component and two exponentially decaying components with tau 1 = 6.14+/- 0.92 ms, tau 2 = 36.5+/- 3.29 ms (S.D) n = 7 for steps to -120 mV. Currents recorded in the outside-out patch configuration were often unexpectedly large and up to 5% of whole-cell currents obtained in the same cell, suggesting an uneven channel distribution in the plasmalemma of Sf-9 cells. The pharmacology of a number of chloride channel blockers, including anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was investigated and showed for the first time that perrhenate is an effective blocker of C1C-1 and that it has a complex mechanism of action. Further, the potency of A9C was found to be dependent on external chloride concentration. As in studies on muscle cells themselves, blockade was rapidly effective and easily reversible, except when applying the indanyloxyacetate derivative, IAA94/95, which took up to 10 min to act, and, consistent with an intracellular site of action, was difficult to reverse by washing. Mutation of the highly conserved arginine at position 304 to a glutamic acid did not significantly alter the behaviour of the channel.

Characterization of the small cryptic plasmid, pIMVS1, of Salmonella enterica ser. Typhimurium.

The nucleotide sequence has been determined of a small cryptic plasmid of Salmonella Typhimurium, designated pIMVS1, which correlated with an outbreak of gastroenteritis. This plasmid has a size of 3357 bp with no known functions. It does not encode any protein products but shows homology to several well-characterized plasmids. The replication system with RNAI and RNAII and the replication origin (oriV) is highly similar to that of plasmid p15A. It also has a putative mobilisation origin similar to that of ColE1.

Comparison of improved BACTEC and Lowenstein-Jensen media for culture of mycobacteria from clinical specimens.

A 4-month trial involving 2,563 routine clinical specimens was conducted to compare the improved BACTEC TB system (12B medium) with the conventional Lowenstein-Jensen (LJ) media for the isolation, identification, and susceptibility testing of mycobacteria. One hundred sixty-two mycobacterial isolates were recovered, 147 (91%) with BACTEC and 118 (73%) with LJ media. Of these, 62 were Mycobacterium tuberculosis complex strains, 59 (95%) of which were isolated with BACTEC and 54 (87%) of which were isolated with LJ media. Of the remaining 100 isolates, which were mycobacteria other than tuberculosis (MOTT), BACTEC and LJ media detected 88 and 64%, respectively. The contamination rate was significantly higher in BACTEC (5%) than in LJ media (3.3%). The mean isolation time for M. tuberculosis complex with BACTEC was 15.5 days, compared with 25.6 days with LJ. For MOTT, the mean isolation times were 5.8 and 21.4 days, respectively. Identification of 32 M. tuberculosis complex isolates and 38 isolates of MOTT by the BACTEC NAP (p-nitro-alpha-acetylamino-beta-hydroxypropiophenone) inhibition test gave 100% agreement with conventional biochemical identifications. The results of susceptibility testing of 18 M. tuberculosis complex isolates with BACTEC agreed completely with those obtained by the resistance ratio method.