Heterogeneity and function of K(ATP) channels in canine hearts.

BACKGROUND: The concept that pore-forming Kir6.2 and regulatory SUR2A subunits form cardiac ATP-sensitive potassium (K(ATP)) channels is challenged by recent reports that SUR1 is predominant in mouse atrial K(ATP) channels. OBJECTIVE: To assess SUR subunit composition of K(ATP) channels and consequence of K(ATP) activation for action potential duration (APD) in dog hearts. METHODS: Patch-clamp techniques were used on isolated dog cardiomyocytes to investigate K(ATP) channel properties. Dynamic current clamp, by injection of a linear K(+) conductance to simulate activation of the native current, was used to study the consequences of K(ATP) activation on APD. RESULTS: Metabolic inhibitor (MI)-activated current was not significantly different from pinacidil (SUR2A-specific)-activated current, and both currents were larger than diazoxide (SUR1-specific)-activated current in both the atrium and the ventricle. Mean K(ATP) conductance (activated by MI) did not differ significantly between chambers, although, within the ventricle, both MI-induced and pinacidil-induced currents tended to decrease from the epicardium to the endocardium. Dynamic current-clamp results indicate that myocytes with longer baseline APDs are more susceptible to injected K(ATP) current, a result reproduced in silico by using a canine action potential model (Hund-Rudy) to simulate epicardial and endocardial myocytes. CONCLUSIONS: Even a small fraction of K(ATP) activation significantly shortens APD in a manner that depends on existing heterogeneity in K(ATP) current and APD.

The pathway for the production of inositol hexakisphosphate in human cells.

The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.

Proprotein processing within secretory dense core granules of Tetrahymena thermophila.

In the ciliate Tetrahymena thermophila, the polypeptides stored in secretory dense core granules (DCGs) are generated by proteolytic processing of precursors, and the mature products assemble as a crystal. Previous observations suggested that this maturation involves precise cleavage at distinct motifs by a small number of enzymes. To test these inferences, we analyzed the determinants for site-specific processing of pro-Grl1p (Granule lattice protein 1) by complete gene replacement with modified alleles. Contrary to the predictions of previous models, none of the component amino acids in a putative processing motif was necessary for targeted cleavage. Indeed, cleavage at a range of alternative positions near the native site was consistent with normal DCG assembly. Furthermore, substitution of other classes of processing site motifs did not perturb DCG structure or function. These results suggest that processing can be catalyzed by multiple proteases, for which substrate accessibility may be the prime determinant of site specificity. Consistent with this, inhibition of either subtilisin or cathepsin family proteases resulted in delayed processing of pro-Grl1p.

The synthesis of inositol hexakisphosphate. Characterization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase.

The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP(6)) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP(6) by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP(5)). Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP(5) 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP(6) or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP(5) to InsP(6)/min/mg of protein (V(max)). The Michaelis-Menten constant for InsP(5) was 0.4 microM and for ATP was 21 microM. Saccharomyces cerevisiae lacking IPK1 do not produce InsP(6) and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP(5) 2-kinase in a yeast ipk1 null strain restored the synthesis of InsP(6) and rescued the gle1-2 ipk1-4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP(5) 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP(6) synthesis in mammalian cells will allow for further studies of the InsP(6) signaling functions.