Cholesterol sensitivity and lipid raft targeting of Kir2.1 channels

This study investigates how changes in the level of cellular cholesterol affect inwardly rectifying K+ channels belonging to a family of strong rectifiers (Kir2). In an earlier study we showed that an increase in cellular cholesterol suppresses endogenous K+ current in vascular endothelial cells, presumably due to effects on underlying Kir2.1 channels. Here we show that, indeed, cholesterol increase strongly suppressed whole-cell Kir2.1 current when the channels were expressed in a null cell line. However, cholesterol level had no effect on the unitary conductance and only little effect on the open probability of the channels. Moreover, no cholesterol effect was observed either on the total level of Kir2.1 protein or on its surface expression. We suggest, therefore, that cholesterol modulates not the total number of Kir2.1 channels in the plasma membrane but rather the transition of the channels between active and silent states. Comparing the effects of cholesterol on members of the Kir2.x family shows that Kir2.1 and Kir2.2 have similar high sensitivity to cholesterol, Kir2.3 is much less sensitive, and Kir2.4 has an intermediate sensitivity. Finally, we show that Kir2.x channels partition virtually exclusively into Triton-insoluble membrane fractions indicating that the channels are targeted into cholesterol-rich lipid rafts.     

Hypercholesterolemia suppresses Kir channels in porcine bone marrow progenitor cells in vivo

OBJECTIVE: Inwardly-rectifying K(+) (Kir) channels are responsible for maintaining membrane potentials in a variety of cell types including endothelial cells where they modulate endothelium-dependent vasorelaxation. The goal of this study is to determine the functional expression of Kir channels in porcine bone marrow-derived side population (BM-SP) cells that demonstrate phenotypes of endothelial progenitor cells (EPCs). We further asses the hypercholesterolemia sensitivity of Kir channels in BM-SP cells, which may play a key role in hypercholesterolemia-mediated regulation of EPCs. METHODS: To assess the effect of hypercholesterolemia on Kir channels in BM-SP, Kir currents were recorded in SP cells sorted from the bone marrow of healthy or hypercholesterolemic animals. RESULTS: We found Kir channels constitute the major conductance in porcine bone marrow-derived side population (BM-SP) cells. These cells are defined by their efficiency of Hoechst dye efflux and have been reported to differentiate into multiple cell lineages including endothelium in vivo. We demonstrate here that porcine BM-SP cells differentiate to an endothelial lineage (CD31(+), vWF(+)) supporting the hypothesis that these cells are endothelial progenitor cells. Also, BM-SP cells express Kir with biophysical properties recapitulating those in mature endothelial cells, but with a much higher current density. Flow cytometric (FACS) analysis indicated that the number of SP cells was unaffected by hypercholesterolemia. However, hypercholesterolemia significantly inhibited Kir channels in BM-SP cells. CONCLUSIONS: We successfully demonstrate that BM side population cells represent an origin of endothelial progenitor cells. This study further shows, for the fist time, that the functional expression of Kir channels in bone marrow (BM)-derived SP. Moreover, we demonstrate that hypercholesterolemia condition significantly suppresses the Kir channels in BM-SP cells, suggesting that hypercholesterolemia-mediated regulation of Kir channels may be an important factor not only in dysfunction of mature endothelium but also in dysfunction of BM-SP cells.     

Enantioselective protein-sterol interactions mediate regulation of both prokaryotic and eukaryotic inward rectifier K+ channels by cholesterol

Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward rectifier K(+) (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by (86)Rb(+) influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket.     

Endosomal KATP channels as a reservoir after myocardial ischemia: a role for SUR2 subunits

ATP-sensitive K(+) (K(ATP)) channels, composed of inward rectifier K(+) (Kir)6.x and sulfonylurea receptor (SUR)x subunits, are expressed on cellular plasma membranes. We demonstrate an essential role for SUR2 subunits in trafficking K(ATP) channels to an intracellular vesicular compartment. Transfection of Kir6.x/SUR2 subunits into a variety of cell lines (including h9c2 cardiac cells and human coronary artery smooth muscle cells) resulted in trafficking to endosomal/lysosomal compartments, as assessed by immunofluorescence microscopy. By contrast, SUR1/Kir6.x channels efficiently localized to the plasmalemma. The channel turnover rate was similar with SUR1 or SUR2, suggesting that the expression of Kir6/SUR2 proteins in lysosomes is not associated with increased degradation. Surface labeling of hemagglutinin-tagged channels demonstrated that SUR2-containing channels dynamically cycle between endosomal and plasmalemmal compartments. In addition, Kir6.2 and SUR2 subunits were found in both endosomal and sarcolemmal membrane fractions isolated from rat hearts. The balance of these K(ATP) channel subunits shifted to the sarcolemmal membrane fraction after the induction of ischemia. The K(ATP) channel current density was also increased in rat ventricular myocytes isolated from hearts rendered ischemic before cell isolation without corresponding changes in subunit mRNA expression. We conclude that an intracellular pool of SUR2-containing K(ATP) channels exists that is derived by endocytosis from the plasma membrane. In cardiac myocytes, this pool can potentially play a cardioprotective role by serving as a reservoir for modulating surface K(ATP) channel density under stress conditions, such as myocardial ischemia.     

Sensitivity of volume-regulated anion current to cholesterol structural analogues

Depletion of membrane cholesterol and substitution of endogenous cholesterol with its structural analogues was used to analyze the mechanism by which cholesterol regulates volume-regulated anion current (VRAC) in endothelial cells. Depletion of membrane cholesterol enhanced the development of VRAC activated in a swelling-independent way by dialyzing the cells either with GTPgammaS or with low ionic strength solution. Using MbetaCD-sterol complexes, 50-80% of endogenous cholesterol was substituted with a specific analogue, as verified by gas-liquid chromatography. The effects of cholesterol depletion were reversed by the substitution of endogenous cholesterol with its chiral analogue, epicholesterol, or with a plant sterol, beta-sitosterol, two analogues that mimic the effect of cholesterol on the physical properties of the membrane bilayer. Alternatively, when cholesterol was substituted with coprostanol that has only minimal effect on the membrane physical properties it resulted in VRAC enhancement, similar to cholesterol depletion. In summary, our data show that these channels do not discriminate between the two chiral analogues of cholesterol, as well as between the two cholesterols and beta-sitosterol, but discriminate between cholesterol and coprostanol. These observations suggest that endothelial VRAC is regulated by the physical properties of the membrane.     

Direct Regulation of Prokaryotic Kir Channel by Cholesterol

Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by ⁸⁶Rb⁺ uptake. Our results show that ⁸⁶Rb⁺ flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses ⁸⁶Rb⁺ flux by >50%, and activity is completely inhibited at 12–15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated ⁸⁶Rb⁺ uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 μg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific.Inwardly rectifying potassium channels (Kir) are known to play critical roles in the regulation of multiple cellular functions including membrane excitability, heart rate, and vascular tone (1–3). Kir channels are classified into seven subfamilies (Kir1–7) identified by distinct biophysical properties and sensitivities to different regulators (2). Our earlier studies have shown that Kir2 channels, one of the major subfamilies of Kir that are responsible for maintaining membrane potential in a variety of cell types, are strongly suppressed by the elevation of membrane cholesterol (4, 5). Cholesterol-induced suppression of Kir2 was first observed in aortic endothelial cells (4), in which resting K⁺ conductance is dominated by Kir2.1 and Kir2.2 channels (6), and then when channels were heterologously expressed in Chinese hamster ovary cells (5, 7). Furthermore, the same effect was observed ex vivo in endothelial cells and bone marrow-derived progenitor cells isolated from hypercholesterolemic pigs (8, 9).In terms of the mechanism, the first insights came from comparing the effects of cholesterol and of its chiral analogue, epicholesterol. Although the two sterols are known to have almost identical effects on the biophysical properties of the lipid bilayer (10, 11), their impact on Kir activity is completely different; partial substitution of endogenous cholesterol with epicholesterol resulted in significant increase in Kir current in endothelial cells (4). These observations suggest that specific sterol-protein interactions may be involved in the cholesterol sensitivity of Kir2 channels. However, in the complex environment of the plasma membrane, cholesterol may interact not only with the channels themselves but also with other proteins, which in turn may regulate the activity of the channels. In the cellular environment, therefore, it is impossible to discriminate between direct channel-cholesterol interactions and indirect effects. Moreover, it is impossible to define the actual concentrations of cholesterol in any given membrane compartment. To quantitatively test direct cholesterol-protein interactions, it is necessary to examine sensitivity of pure Kir channels to membrane cholesterol in a membrane of defined lipid composition. To date, only the cytoplasmic domains of several mammalian Kir channels have been purified (Kir2.1, Kir3.1, and Kir3.2) (12–15). We therefore concentrate in this study on the effect of cholesterol on two bacterial K⁺ channels that differ in the level of their homology to mammalian Kir channels, KirBac1.1 and KcsA. KirBac channels have high sequence homology with mammalian Kirs (e.g. 52% homology between KirBac1.1 and Kir2.1; see Fig. 7A) and have now been extensively used as structural models of mammalian Kir channels (3, 16, 17). The sequence similarity between KcsA and mammalian K channels lies mainly in the transmembrane domain (18). The overall sequence homology of KcsA to mammalian Kir channels is relatively low (e.g. 22% homology between KcsA and Kir2.1; see Fig. 7A), with an entirely different cytoplasmic domain structure.Open in a separate windowFIGURE 7.Cholesterol has no effect on KcsA-mediated ⁸⁶Rb⁺ uptake. A, time courses of ⁸⁶Rb⁺ uptake into liposomes reconstituted with 50 μg of cholesterol/mg of PL and as compared with liposomes containing no cholesterol (control). Both batches of liposomes contained 5 μg of KcsA/mg of PL. Blank liposomes contain no protein. The points represent averages of three independent experiments (means ± S.D.). B, normalized time courses of ⁸⁶Rb⁺ uptake in liposomes incorporating 50, 150, and 250 μg of cholesterol/mg of PL. C, maximal uptake of ⁸⁶Rb⁺ after 240 s in liposomes containing 10, 25, 50, 100, 150, 200, and 250 μg of cholesterol/mg of PL normalized to control (means ± S.D. of 3–5 independent experiments; *, p < 0.05). DPM, disintegrations per minute.Here we show that, similarly to Kir2 channels, prokaryotic Kir channels incorporated into liposomes are strongly suppressed by an increase in membrane cholesterol. Furthermore, the sensitivity of prokaryotic Kir to cholesterol is stereo-selective to cholesterol optical analogues. In contrast, KscA channels are insensitive to membrane cholesterol. These observations suggest that cholesterol directly suppresses Kir channels.     

Specificity of cholesterol and analogs to modulate BK channels points to direct sterol-channel protein interactions

The activity (Po) of large-conductance voltage/Ca(2+)-gated K(+) (BK) channels is blunted by cholesterol levels within the range found in natural membranes. We probed BK channel-forming α (cbv1) subunits in phospholipid bilayers with cholesterol and related monohydroxysterols and performed computational dynamics to pinpoint the structural requirements for monohydroxysterols to reduce BK Po and obtain insights into cholesterol's mechanism of action. Cholesterol, cholestanol, and coprostanol reduced Po by shortening mean open and lengthening mean closed times, whereas epicholesterol, epicholestanol, epicoprostanol, and cholesterol trisnorcholenic acid were ineffective. Thus, channel inhibition by monohydroxysterols requires the β configuration of the C3 hydroxyl and is favored by the hydrophobic nature of the side chain, while having lax requirements on the sterol A/B ring fusion. Destabilization of BK channel open state(s) has been previously interpreted as reflecting increased bilayer lateral stress by cholesterol. Lateral stress is controlled by the sterol molecular area and lipid monolayer lateral tension, the latter being related to the sterol ability to adopt a planar conformation in lipid media. However, we found that the differential efficacies of monohydroxysterols to reduce Po (cholesterol≥coprostanol≥cholestanol>epicholesterol) did not follow molecular area rank (coprostanol>epicholesterol>cholesterol>cholestanol). In addition, computationally predicted energies for cholesterol (effective BK inhibitor) and epicholesterol (ineffective) to adopt a planar conformation were similar. Finally, cholesterol and coprostanol reduced Po, yet these sterols have opposite effects on tight lipid packing and, likely, on lateral stress. Collectively, these findings suggest that an increase in bilayer lateral stress is unlikely to underlie the differential ability of cholesterol and related steroids to inhibit BK channels. Remarkably, ent-cholesterol (cholesterol mirror image) failed to reduce Po, indicating that cholesterol efficacy requires sterol stereospecific recognition by a protein surface. The BK channel phenotype resembled that of α homotetramers. Thus, we hypothesize that a cholesterol-recognizing protein surface resides at the BK α subunit itself.     

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane

The ATP-sensitive K(+) (K(ATP)) channels in both sarcolemmal (sarcK(ATP)) and mitochondrial inner membrane (mitoK(ATP)) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcK(ATP) contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoK(ATP) remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing K(ATP) into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing K(ATP) channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing K(ATP) channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional K(ATP) channels in mitochondria was significantly increased by PMA, as shown by K(ATP)-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing K(ATP) channels was blocked by a selective PKCepsilon inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the K(ATP) channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKCepsilon. PKC isoform-regulated mitochondrial import of K(ATP) channels may have significant implication in cardioprotection of IPC.     

Calcium-sensing receptor decreases cell surface expression of the inwardly rectifying K+ channel Kir4.1

The Ca(2+)-sensing receptor (CaR) regulates salt and water transport in the kidney as demonstrated by the association of gain of function CaR mutations with a Bartter syndrome-like, salt-wasting phenotype, but the precise mechanism for this effect is not fully established. We found previously that the CaR interacts with and inactivates an inwardly rectifying K(+) channel, Kir4.1, which is expressed in the distal nephron that contributes to the basolateral K(+) conductance, and in which loss of function mutations are associated with a complex phenotype that includes renal salt wasting. We now find that CaR inactivates Kir4.1 by reducing its cell surface expression. Mutant CaRs reduced Kir4.1 cell surface expression and current density in HEK-293 cells in proportion to their signaling activity. Mutant, activated Gα(q) reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via Gα(i) and Gα(q). Other α subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of Gα(q) on Kir4.1, whereas knockdown of the clathrin heavy chain had no effect. CaR had no comparable effect on the renal outer medullary K(+) channel, an apical membrane distal nephron K(+) channel that is internalized by clathrin-coated vesicles. Co-immunoprecipitation studies showed that the CaR and Kir4.1 physically associate with caveolin-1 in HEK cells and in kidney extracts. Thus, the CaR decreases cell surface expression of Kir4.1 channels via a mechanism that involves Gα(q) and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.     

Activation of a small-conductance Ca(2+)-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells.

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.     

The Stoichiometry of Gbeta gamma binding to G-protein-regulated inwardly rectifying K+ channels (GIRKs)

G-protein-coupled inwardly rectifying K(+) (GIRK; Kir3.x) channels are the primary effectors of numerous G-protein-coupled receptors. GIRK channels decrease cellular excitability by hyperpolarizing the membrane potential in cardiac cells, neurons, and secretory cells. Although direct regulation of GIRKs by the heterotrimeric G-protein subunit Gbetagamma has been extensively studied, little is known about the number of Gbetagamma binding sites per channel. Here we demonstrate that purified GIRK (Kir 3.x) tetramers can be chemically cross-linked to exogenously purified Gbetagamma subunits. The observed laddering pattern of Gbetagamma attachment to GIRK4 homotetramers was consistent with the binding of one, two, three, or four Gbetagamma molecules per channel tetramer. The fraction of channels chemically cross-linked to four Gbetagamma molecules increased with increasing Gbetagamma concentrations and approached saturation. These results suggest that GIRK tetrameric channels have four Gbetagamma binding sites. Thus, GIRK (Kir 3.x) channels, like the distantly related cyclic nucleotide-gated channels, are tetramers and exhibit a 1:1 subunit/ligand binding stoichiometry.     

Evidence against functional heteromultimerization of the KATP channel subunits Kir6.1 and Kir6.2

K(ATP) channels consist of pore-forming potassium inward rectifier (Kir6.x) subunits and sulfonylurea receptors (SURs). Although Kir6.1 or Kir6.2 coassemble with different SUR isoforms to form heteromultimeric functional K(ATP) channels, it is not known whether Kir6.1 and Kir6.2 coassemble with each other. To define the molecular identity of K(ATP) channels, we used adenoviral gene transfer to express wild-type and dominant-negative constructs of Kir6.1 and Kir6.2 in a heterologous expression system (A549 cells) and in native cells (rabbit ventricular myocytes). Dominant-negative (DN) Kir6.2 gene transfer suppressed current through heterologously expressed SUR2A + Kir6.2 channels. Conversely, DN Kir6.1 suppressed SUR2B + Kir6.1 current but had no effect on coexpressed SUR2A + Kir6. 2. We next probed the ability of Kir6.1 and Kir6.2 to affect endogenous K(ATP) channels in adult rabbit ventricular myocytes, using adenoviral vectors to achieve efficient gene transfer. Infection with the DN Kir6.2 virus for 72 h suppressed pinacidil-inducible K(ATP) current density measured by whole-cell patch clamp. However, there was no effect of infection with the DN Kir6.1 on the K(ATP) current. Based on these functional assays, we conclude that Kir6.1 and Kir6.2 do not heteromultimerize with each other and that Kir6.2 is the sole K(ATP) pore-forming subunit in the surface membrane of heart cells.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

ATP-sensitive K(+) channels regulate the concentrative adenosine transporter CNT2 following activation by A(1) adenosine receptors

This study describes a novel mechanism of regulation of the high-affinity Na(+)-dependent adenosine transporter (CNT2) via the activation of A(1) adenosine receptors (A(1)R). This regulation is mediated by the activation of ATP-sensitive K(+) (K(ATP)) channels. The high-affinity Na(+)-dependent adenosine transporter CNT2 and A(1)R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (-)-N(6)-(2-phenylisopropyl)adenosine is fully inhibited by K(ATP) channel blockers and mimicked by a K(ATP) channel opener. A(1)R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A(1)R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A(1)R, CNT2, and K(ATP) channels. These results also suggest that the activation of K(ATP) channels under metabolic stress can be mediated by the activation of A(1)R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for K(ATP) channels.     

Regulation of cardiac inwardly rectifying potassium channels by membrane lipid metabolism

Types and distributions of inwardly rectifying potassium (Kir) channels are one of the major determinants of the electrophysiological properties of cardiac myocytes. Kir2.1 (classical inward rectifier K(+) channel), Kir6.2/SUR2A (ATP-sensitive K(+) channel) and Kir3.1/3.4 (muscarinic K(+) channels) in cardiac myocytes are commonly upregulated by a membrane lipid, phosphatidylinositol 4,5-bisphosphates (PIP(2)). PIP(2) interaction sites appear to be conserved by positively charged amino acid residues and the putative alpha-helix in the C-terminals of Kir channels. PIP(2) level in the plasma membrane is regulated by the agonist stimulation. Kir channels in the cardiac myocytes seem to be actively regulated by means of the change in PIP(2) level rather than by downstream signal transduction pathways.     

Relationship between Kir2.1/Kir2.3 activity and their distributions between cholesterol-rich and cholesterol-poor membrane domains

Our earlier studies have shown that Kir2.x channels are suppressed by an increase in the level of cellular cholesterol, whereas cholesterol depletion enhances the activity of the channels. In this study, we show that Kir2.1 and Kir2.3 channels have double-peak distributions between cholesterol-rich (raft) and cholesterol-poor (non-raft) membrane fractions, indicating that the channels exist in two different types of lipid environment. We also show that whereas methyl--cyclodextrin-induced cholesterol depletion removes cholesterol from both raft and non-raft membrane fractions, cholesterol enrichment results in cholesterol increase exclusively in the raft fractions. Kinetics of both depletion-induced Kir2.1 enhancement and enrichment-induced Kir2.1 suppression correlate with the changes in the level of raft cholesterol. Furthermore, we show not only that cholesterol depletion shifts the distribution of the channels from cholesterol-rich to cholesterol-poor membrane fractions but also that cholesterol enrichment has the opposite effect. These observations suggest that change in the level of raft cholesterol alone is sufficient to suppress Kir2 activity and to facilitate partitioning of the channels to cholesterol-rich domains. Therefore, we suggest that partitioning to membrane rafts plays an important role in the sensitivity of Kir2 channels to cholesterol. ion channels; inward rectifiers; inwardly rectifying potassium channels     

Polyamine permeation and rectification of Kir4.1 channels

Inward rectifier K(+) (Kir) channels are expressed in multiple neuronal and glial cells. Recent studies have equated certain properties of exogenously expressed Kir4.1 channels with those of native K(+) currents in brain cells, as well as demonstrating the expression of Kir4.1 subunits in these tissues. There are nagging problems however with assigning native currents to Kir4.1 channels. One major concern is that in many native tissues, the putatively correlated currents show much weaker rectification than typically reported for cloned Kir4.1 channels. We have now examined the polyamine-dependence of Kir4.1 channels expressed at high density in Cosm6 cells, using inside-out membrane patches. The experiments reveal a complex and variable rectification that can help explain the variability reported for candidate Kir4.1 currents in native cells. Most importantly, rectification seems to be incomplete, even at high polyamine concentrations. In excised membrane patches, with high levels of expression, and high concentrations of spermine, there is approximately 15% residual conductance that is insensitive to spermine. From a biophysical perspective, this is a striking finding, and indicates either that a bound spermine fails to completely block permeation or that significant spermine permeation (i.e. 'punchthrough') is occurring. To examine this further, we have examined block by philanthotoxin (PhTx, essentially spermine with a bulky tail). PhTx block, while less potent, is more complete than spermine block. This leads us to propose that spermine 'punchthrough' may be significant in Kir4 channels, and that this may be a major contributor to the weak rectification observed under physiological conditions.