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

STOCs resulted from BKCa activity and were dependent on extracellular Ca2+ but not significantly on voltage-dependent Ca2+ channels. STOCs were dependent on intracellular Na+ and intracellular calcium store refilling state. We suggest that Ca2+ entry into the cell through reverse-mode Na+/Ca2+ exchange determines calcium store refilling, which in turn regulates STOC generation in human coronary VSMCs.

The bilayer-couple model predicts a reversible membrane crenation for an increasing ratio of external to internal monolayer area. This was comprehensively proven. However, individual erythrocytes may undergo dramatic shape changes within seconds when the suspension medium is changed. In contrast, under physiological conditions with no addition of membrane active compounds, active phospholipid translocation and passive flip-flops are comparatively slow. We propose that conformational changes of the anion-exchange protein, band 3, may rapidly alter the monolayer area ratio. Band 3 occupies about 10% of the total membrane area of human erythrocytes. Under physiological conditions, its conformers are asymmetrically distributed with about 90% of the transport sites facing the cytoplasm. This distribution is altered when external conformations are recruited by changing the transmembranous Cl- gradient, the external pH, or by the application of inhibitors. In experiments, recruitment by low ionic strength caused a rapid, temporary formation of echinocytes. This suspension effect could also be found at high ionic concentrations, when Cl- was replaced by SO4(2-). Inhibitors known to recruit the external band 3 conformation, like DIDS, SITS and flufenamic acid, are echinocytogenic. For inhibitors not recruiting a certain conformation, e.g. phenylglyoxal and niflumic acid, no shape effect was found. Since band 3 ensures a fast equilibrium of internal and external anions these ions are usually distributed according to the transmembrane potential (TMP). In the literature, a correlation of TMP and band 3 conformation, as well as a correlation of TMP and red cell shape, is described. In the proposed model, low external Cl- concentrations, inhibitors, or a negative TMP may recruit the transport sit outwards.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract-We recently demonstrated that farnesol, a 15-carbon isoprenoid, blocks L-type Ca 2ϩ channels in vascular smooth muscle cells. To elucidate farnesolЈs mechanism of action, we performed whole-cell and perforated-patch clamp experiments in rat aortic A7r5 cells and in Chinese hamster ovary (CHO) C9 cells expressing smooth muscle Ca 2ϩ channel ␣ 1C subunits. Farnesol dose-dependently and voltage-independently inhibited Ba 2ϩ currents in both A7r5 and CHOC9 cells, with similar half-maximal inhibitions at 2.6 and 4.3 mmol/L, respectively (PϭNS). In both cell lines, current inhibition by farnesol was prominent over the whole voltage range without changes in the current-voltage relationship peaks. Neither intracellular infusion of the stable GDP analogue guanosine-5Ј-O-(2-thiodiphosphate) (100 mmol/L) via the patch pipette nor strong conditioning membrane depolarization prevented the inhibitory effect of farnesol, which indicates G protein-independent inhibition of Ca 2ϩ channels. In an analysis of the steady-state inactivation curve for voltage dependence, farnesol induced a significant, negative shift (Ϸ10 mV) of the potential causing 50% channel inactivation in both cell lines (PϽ0.001). In contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unlike pharmacological Ca 2ϩ channel blockers, farnesol blocked Ca 2ϩ currents in the resting state: initial block was 63Ϯ8% in A7r5 cells and 50Ϯ9% in CHOC9 cells at a holding potential of Ϫ80 mV. We then gave 500 mg/kg body weight farnesol by gavage to Sabra hypertensive and normotensive rats and found that farnesol reduced blood pressure significantly in the hypertensive strain for at least 48 hours. We conclude that farnesol may represent an endogenous smooth muscle L-type Ca 2ϩ channel antagonist. Because farnesol is active in cells expressing only the pore-forming ␣ 1 subunit, the data further suggest that this subunit represents the molecular target for farnesol binding and principal action. Key Words: smooth muscle cells Ⅲ farnesol Ⅲ patch clamp Ⅲ calcium channel blockers Ⅲ L-class channels F arnesol is 1 of several nonsterol mevalonate derivatives in the cholesterol pathway. 1 Farnesol is derived from farnesyl pyrophosphate, a C 15 isoprenoid lipid implicated in the regulation of G protein activity. 2 Some studies suggest that farnesol participates in the control of cholesterol synthesis by increasing 3-hydroxy-3-methylglutaryl coenzyme A reductase degradation. 3,4 Others suggest that farnesol is implicated in the regulation of cell growth. 5 Roullet et al 6 recently showed that farnesol inhibited contraction in both human and animal arteries. This effect explained earlier reports of a mevalonate-dependent positive regulation of vascular tone. 7,8 These studies featured a series of experiments that showed that farnesol blunted the KCl-induced increase in intracellular Ca 2ϩ concentration in intact arteries and vascular smooth muscle cells (VSMCs) in culture. 9 The patch-clamp technique revealed that micromolar co...

We investigated pinacidil-activated K+ currents in vascular smooth muscle cells (VSMC) from human coronary arteries with the patch-clamp method. In 19 of 54 VSMC, pinacidil (1 and 20 microM) induced a large, nonrectifying, outward current [IK(ATP)] and increased voltage-dependent outward K+ currents [IK(Ca)] positive to voltages of -25 mV. The pinacidil-induced (1 microM) IK(ATP) was blocked by glibenclamide (3 microM) but was not affected by iberiotoxin (100-300 nM). Pinacidil activated up to 150 functionally active ATP-dependent K+ channels (KATP channels) per cell with a single-channel conductance of approximately 17 pS at physiological membrane potentials (between -80 and -30 mV) and K+ gradients (6 mM/130 mM). In 26 of 54 VSMC, on the other hand, pinacidil (1-20 microM) failed to induce IK(ATP) but increased IK(Ca). This current was completely blocked by iberiotoxin (100-300 nM) and tetraethylammonium (1 mM) but not by glibenclamide (3 microM). The single-channel conductance of the channel underlying IK(Ca) was approximately 150 +/- 16 pS between -10 and +30 mV, consistent with large-conductance, maxi Ca(2+)-activated, K+ channels (BKCa channels). We conclude that pinacidil is a nonselective K+ channel opener targeting KATP and BKCa channels. Furthermore, the conductance of KATP channels in human coronary arteries is likely to be small under physiological conditions.