A strain of C. difficile that was resistant to fluoroquinolones and had binary toxin and a partial deletion of the tcdC gene was responsible for this outbreak of C. difficile-associated diarrhea. Exposure to fluoroquinolones or cephalosporins was a risk factor.

A new method is described as an alternative to whole-cell recording in order to prevent "wash-out" of the muscarinic response to acetylcholine (ACh) in rat lacrimal gland cells. The membrane of a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without the loss or aheration of cytoplasmic compounds necessary for the maintenance of the response to ACh. With normal whole-cell recording in these cells, the response to ACh, seen as the activation of Ca-activated K and CI currents, lasts for ~5 rain. With the nystatin method, the response is not diminished after 1 h. Nystatin, applied extracellularly, is shown to cause a rapid and reversible increase of membrane conductance to cations. In the absence of wash-out, we were able to obtain dose-response curves for the effect of ACh on Ca-activated K currents. An increase of [ACh] caused an increase in the K current, with apparent saturation at concentrations above ~ 1 #M ACh. The delay between ACh application and the activation of K current was inversely related to [ACh] and reached a minimum value of 0.7-1.0 s at high [ACh].

Voltage-dependent movement of a sodium channel S4 segment was examined by cysteine scanning mutagenesis and testing accessibility of the residues to hydrophilic cysteine-modifying reagents. These experiments indicate that 2 charged S4 residues move completely from an internally accessible to an externally accessible location in response to depolarization by passage through a short "channel" in the protein. The energetic problems of S4 movement have thus been solved in the same way that may ion channels achieve highly selective and rapid ion permeation through an open pore, by restricting the contact region between the permion and its channel.

No abstract

The principal voltage-sensitive sodium channel from human heart has been cloned, sequenced, and functionally expressed. The cDNA, designated hH1, encodes a 2016-amino acid protein that is homologous to other members of the sodium channel multigene family and bears >90% identity to the tetrodotoxin-insensitive sodium channel characteristic of rat heart and of immature and denervated rat skeletal muscle. Northern blot analysis demonstrates an =9.0-kilobase transcript expressed in human atrial and ventricular cardiac muscle but not in adult skeletal muscle, brain, myometrium, liver, or spleen. When expressed in Xenopus oocytes, hHl exhibits rapid activation and inactivation kinetics similar to native cardiac sodium channels. The single channel conductance of hHl to sodium ions is about twice that of the homologous rat channel and hHl is more resistant to block by tetrodotoxin (ICso = 5.7 pM). hHl is also resistant to Iu-conotoxin but sensitive to block by therapeutic concentrations of lidocaine in a use-dependent manner.

No abstract

The mutation R1448C substitutes a cysteine for the outermost arginine in the fourth transmembrane segment (S4) of domain 4 in skeletal muscle sodium channels. We tested the accessibility of this cysteine residue to hydrophilic methanethiosulfonate reagents applied to the extracellular surface of cells expressing these mutant channels. The reagents irreversibly increase the rate of inactivation of R1448C, but not wild-type, channels. Cysteine modification is voltage dependent, as if depolarization extends this residue into the extracellular space. The rate of cysteine modification increases with depolarization and has the voltage dependence and kinetics expected for the movement of a voltage sensor controlling channel gating.

Sodium channels have four homologous domains (D1-D4) each with six putative transmembrane segments (S1-$6). The highly charged $4 segments in each domain are postulated voltage sensors for gating. We made 15 charge-neutralizing or -reversing substitutions in the first or third basic residues (arginine or lysine) by replacement with histidine, glutamine, or glutamate in $4 segments of each domain of the human heart Na ÷ channel. Nine of the mutations cause shifts in the conductance-voltage (G-V) midpoints, and all but two significantly decrease the voltage dependence of peak Na + current, consistent with a role of $4 segments in activation. The decreases in voltage dependence of activation were equivalent to a decrease in apparent gating charge of 0.5-2.1 elementary charges (%) per channel for single charge-neutralizing mutations. Three charge-reversing mutations gave decreases of 1.2-1.9 eo per channel in voltage dependence of activation. The steady-state inactivation (h~) curves were fit by single-component Boltzmann functions and show significant decreases in slope for 9 of the 15 mutants and shifts of midpoints in 9 mutants. The voltage dependence of inactivation time constants is markedly decreased by mutations only in $4D4, providing further evidence that this segment plays a unique role in activation-inactivation coupling.