The mechanism of verapamil block of the delayed rectifier K currents (I K(DR) ) in chick dorsal root ganglion (DRG) neurons was investigated using the whole‐cell patch clamp configuration. In particular we focused on the location of the blocking site, and the active form (neutral or charged) of verapamil using the permanently charged verapamil analogue D890. Block by D890 displayed similar characteristics to that of verapamil, indicating the same state‐dependent nature of block. In contrast with verapamil, D890 was effective only when applied internally, and its block was voltage dependent (136 mV/e‐fold change of the on rate). Given that verapamil block is insensitive to voltage ( Trequattrini et al ., 1998 ), these observations indicate that verapamil reaches its binding site in the uncharged form, and accesses the binding domain from the cytoplasm. In external K and saturating verapamil we recorded tail currents that did not decay monotonically but showed an initial increase (hook). As these currents can only be observed if verapamil unblock is significantly voltage dependent, it has been suggested ( DeCoursey, 1995 ) that neutral drug is protonated upon binding. We tested this hypothesis by assessing the voltage dependence of the unblock rate from the hooked tail currents for verapamil and D890. The voltage dependence of the off rate of D890, but not of verapamil, was well described by adopting the classical Woodhull (1973) model for ionic blockage of Na channels. The voltage dependence of verapamil off rate was consistent with a kinetic scheme where the bound drug can be protonated with rapid equilibrium, and both charged and neutral verapamil can unbind from the site, but with distinct kinetics and voltage dependencies. British Journal of Pharmacology (1999) 126 , 1699–1706; doi: 10.1038/sj.bjp.0702477
An element controlling chloramphenicol resistance (chl) was detected in Streptomyces coelicolor A3(2). Strains sensitive to 1 microgram chloramphenicol ml-1 were obtained among dark scarlet variants. Transfer of the resistance factor was attempted in matings between chloramphenicol-resistant (Chl+) and chloramphenicol-sensitive (Chl-) strains, both of which lacked the SCP1 fertility factor. Transfer of chl was obtained at a much higher rate than that expected for chromosomal markers in SCP1- X SCP1- matings. However, in these particular crosses the latter was also several times higher than usual. All recombinants for chromosomal markers were Chl+. Attempts to locate the chl element failed to distinguish between a chromosomal and an extrachromosomal site. The observed increase in the recombination frequency for chromosomal markers suggests that the chl element may promote recombination.
The effects of verapamil and related phenylalkylamines on neuronal excitability were investigated in isolated neurons of rat intracardiac ganglia using whole-cell perforated patch-clamp recording. Verapamil (>/=10 microM) inhibits tonic firing observed in response to depolarizing current pulses at 22 degrees C. The inhibition of discharge activity is not due to block of voltage-dependent Ca2+ channels because firing is not affected by 100 microM Cd2+. The K+ channel inhibitors charybdotoxin (100 nM), 4-aminopyridine (0.5 mM), apamin (30-100 nM), and tetraethylammonium ions (1 mM) also have no effect on firing behavior at 22 degrees C. Verapamil does not antagonize the acetylcholine-induced inhibition of the muscarine-sensitive K+ current (M-current) in rat intracardiac neurons. Verapamil inhibits the delayed outwardly rectifying K+ current with an IC50 value of 11 microM, which is approximately 7-fold more potent than its inhibition of high voltage-activated Ca2+ channel currents. These data suggest that verapamil inhibits tonic firing in rat intracardiac neurons primarily via inhibition of delayed outwardly rectifying K+ current. Verapamil inhibition of action potential firing in intracardiac neurons may contribute, in part, to verapamil-induced tachycardia.
