Na+ channel inactivation, a critical determinant of refractoriness, differs in cardiomyocytes and neurons. In rat brain type IIa (rB2a) Na+ channels, a critical residue in the cytoplasmic linker between domains III and IV regulates fast inactivation such that a Phe-->Gln substitution (F1489Q) inhibits inactivation by at least 85%. Since this residue is conserved in voltage-gated Na+ channels, we tested whether F1485Q, the analogous mutation in human heart (hH1a) Na+ channels, has a similar functional effect. We found that fast inactivation in wild-type (WT) channels expressed in Xenopus oocytes was complete within 15 milliseconds at a test potential of 0 mV, and its time course was biexponential with time constants of 0.4 and 2 milliseconds. But in contrast to rB2a, the FQ mutation inhibited inactivation by < 50% and increased mean single-channel open time by only twofold. Residual fast inactivation was monoexponential, with a time constant similar to that of the slower phase of normal inactivation (2 milliseconds). In the mutant channels, unlike WT, null tracings were absent at holding potentials in the range of -140 to -120 mV, and the voltage range of steady-state inactivation coincided exactly with that of activation, suggesting that residual inactivation was tightly coupled to the open state. As in rB2a, simultaneous mutations of I1484Q and M1486Q, in addition to mutation F1485Q, completely inhibited fast inactivation. Our results show that in heart Na+ channels, the IFM cluster controls the stability of both open- and closed-channel inactivation in a manner qualitatively similar to that in the brain. Structural differences in the putative inactivation receptor may explain the distinct gating patterns in channel subtypes.

5'-Phosphoribosyl-5-aminoimidazole (AIR) carboxylase (EC 4.1.1.21) catalyzes step 6, the carboxylation of AIR to 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid, in the de novo biosynthesis of purine nucleotides. As deduced from the DNA sequence of restriction fragments encoding AIR carboxylase and supported by maxicell analyses, AIR carboxylase was found to be composed of two nonidentical subunits. In agreement with established complementation data, the catalytic subunit (deduced Mr, 17,782) was encoded by the purE gene, while the CO2-binding subunit (deduced Mr, 39,385) was encoded by the purK gene. These two genes formed an operon in which the termination codon of the purE gene overlapped the initiation codon of the purK gene. The 5' end of the purEK mRNA was determined by mung bean nuclease mapping and was located 41 nucleotides upstream of the proposed initiation codon. The purEK operon is regulated by the purR gene product, and a purR regulatory-protein-binding site related to the sequences found in other pur loci was identified in the purEK operon control region.

We have constructed a series of lacZY gene fusion cassettes containing KanR to create active P-galactosidase fusions in anK desired reading frame. The cassettes were created by inserting the Tn5 Kan gene into the unique NruI site present in lackt (1) of the lacZY fusion plasmids described by

IMP dehydrogenase, the product of the guaB locus in Escherichia coli K12, catalyzes the synthesis of XMP by the NAD+ dependent oxidation of IMP. The guaB locus has been subcloned from the Clarke and Carbon plasmid pLC34-10. The sequence of the guaB structural gene and surrounding DNA was determined by the dideoxy chain termination method of Sanger. The 1.533 kb guaB gene encodes an IMP dehydrogenase subunit of molecular weight 54,512. S1 nuclease mapping placed the site of guaBA mRNA initiation approximately 188 bp from the start of the guaB structural gene. The -10 and -35 regions that define the guaBA promoter were located upstream of the start of the guaBA transcription initiation site. The control region of approximately 188 bp does not show any obvious potential for secondary structure. A secondary lambda att site has been identified 42 bp distal to the guaB start codon.