Three types of electrically-operated calcium channels have been identified in the membrane of cultured dorsal root ganglion neurons from mouse embryos using patch clamp technique. Low-threshold inactivating (LTI) channels with the lowest unitary conductance (5.7 pS with 60 mM Sr2+ or 7.2 pS with 60 mM Ba2+) preserved their activity for a long time on excised membrane patches and were insensitive to dihydropyridine Ca channel agonist Bay K8644. Corresponding whole-cell current could be decreased by 40% with 25 microM D-600. High-threshold inactivating (HTI) channels had somewhat higher unitary conductance (7 pS and 11.4 pS for Sr2+ or Ba2+ at 60 mM concentration) and were much more dependent upon intracellular metabolic support. D-600 inhibited the corresponding HTI whole-cell current by about 10%. High-threshold non-inactivating (HTN) channels were the most conducting ones (9 pS and 18.4 pS for 60 mM Sr2+ or 60 mM Ba2+) and their functioning was strongly metabolic dependent. Whole-cell HTN current could be slightly enhanced by 10 microM Bay K8644 due to some prolongation of channel mean open time. Effect of Bay K8644 was much less pronounced than that reported for cardiac cells. HTN whole-cell current could be almost completely blocked by 25 microM D-600. The described three types of calcium channels revealed different potential dependence and absolute values of mean channel open time.

SummaryWe report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8-kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis. Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-terminal oligomerization and arginine-binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0-kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC-lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half-life of approximately 30 min at 90ЊC, whereas B. subtilis AhrC was largely inactivated at 65ЊC. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85ЊC, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus. This pronounced resistance of the repressor-operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.