Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that function both as signal transducers downstream of cell-surface receptors and in constitutive intracellular membrane and protein trafficking pathways. All PI3Ks are dual-specificity enzymes with a lipid kinase activity which phosphorylates phosphoinositides at the 3-hydroxyl, and a protein kinase activity. The products of PI3K-catalysed reactions, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), PtdIns(3,4)P2 and PtdIns(3)P, are second messengers in a variety of signal transduction pathways, including those essential to cell proliferation, adhesion, survival, cytoskeletal rearrangement and vesicle trafficking. Here we report the 2.2 A X-ray crystallographic structure of the catalytic subunit of PI3Kgamma, the class I enzyme that is activated by heterotrimeric G-protein betagamma subunits and Ras. PI3Kgamma has a modular organization centred around a helical-domain spine, with C2 and catalytic domains positioned to interact with phospholipid membranes, and a Ras-binding domain placed against the catalytic domain where it could drive allosteric activation of the enzyme.

Despite intensive interest in understanding the differentiation of vascular smooth muscle cells (VSMC), no information is available about differential regulation of ion channels in these cells. Since expression of the L-type Ca2+ channel can be influenced by differentiation in other cell types, we tested the hypothesis that the L-type (C class) channel is a specific differentiation marker of VSMC and that expression of these channels depends on the state of cell differentiation. We used rat aortic (A7r5) VSMC, which express functional L-type Ca2+ channels, and induced dedifferentiation by cell culture in different media. Treatment with retinoic acid was used to redifferentiate the VSMC. We characterized the differentiated state of the cells by using immunohistochemistry and Western blot analysis for smooth muscle (SM) alpha-actin and SM-myosin heavy chain (MHC). The number of functional Ca2+ channels was significantly decreased in dedifferentiated VSMC and increased upon differentiation with retinoic acid. Ca2+ channel function was assessed by whole-cell voltage clamp techniques. Using Western blot and dihydropyridine binding analysis, we found that the expression of the Ca2+ channel alpha1 subunit, and to a lesser extent the beta2 subunit, was directly correlated with the expression of SM alpha-actin and SM-MHC. We conclude that expression of L-type Ca2+ channel alpha1 subunits, and thus a functional Ca2+ channel, is highly coordinated with expression of the SM-specific proteins required for specialized smooth muscle cell functions. Furthermore, our results demonstrate that the L-type Ca2+ channel is a novel marker for differentiation of VSMC. The data suggest that regulation of ion channel expression during differentiation may have physiological importance for normal smooth muscle function and may influence VSMC behavior under pathophysiological conditions.

K+ channels and their currents are important in vascular tone regulation and are potential therapeutic targets; however, K+ channels in human coronary artery vascular smooth muscle cells (VSMCs) have received little attention. We examined K+ currents in freshly isolated VSMCs from human coronary arteries (n=368 from 32 human hearts) with conventional patch-clamp or perforated-patch techniques with nystatin. We detected four different K+ currents: (1) the delayed rectifier K+ current, IK(dr); (2) the Ca2+-activated K+ current, IK(Ca); (3) the nonrectifying noninactivating outward ATP-dependent K+ current, IK(ATP); and (4) the spontaneous transient outward K+ current, IK(STOC). K+ channels underlying spontaneous transient outward currents probably represent a single clustered population of Ca2+-activated K+ channels functionally associated with Ca2+ release channels in the sarcoplasmic reticulum. Inwardly rectifying K+ currents were not observed. K+ currents were unevenly distributed in that they were not uniformly exhibited by all cells. The most prominent K+ currents were IK(Ca) (100%) and IK(dr) (46%). IK(STOC)s, which have not been previously described in humans, were present in 67% of VSMCs. IK(ATP) was small under physiological conditions; however, IK(ATP) increased markedly after cell stimulation with exogenous or endogenous coronary vasodilators. Thus, IK(ATP) may be particularly relevant in ischemia and could be of special importance as a therapeutic target. We conclude that human coronary VSMCs have unique K+ currents that differ sufficiently from those of other species, thus making the investigation of human material clinically relevant. The findings suggest potential avenues for further therapeutic research.

Background-Hydrogen peroxide (H 2 O 2 ) and reactive oxygen species are implicated in inflammation, ischemiareperfusion injury, and atherosclerosis. The role of ion channels has not been previously explored. Methods and Results-K ϩ currents and membrane potential were recorded in endothelial cells by voltage-and current-clamp techniques. H 2 O 2 elicited both hyperpolarization and depolarization of the membrane potential in a concentration-dependent manner. Low H 2 O 2 concentrations (0.01 to 0.25 mol/L) inhibited the inward-rectifying K