Gossypol Activates Pancreatic Polyamine Catabolism in Normal Rats and Induces Acute Pancreatitis in Transgenic Rats Over-expressing Spermidine/Spermine N 1 -Acetyltransferase

Background: The male antifertility agent gossypol has been reported to induce spermidine/spermine N 1 -acetyltransferase (SSAT) in canine prostate cells. As SSAT is the rate-controlling enzyme in the catabolism of the polyamines and is involved in the development of acute pancreatitis in a recent transgenic rat model, we exposed normal and transgenic rats over-expressing SSAT to gossypol to evaluate its effect on pancreatic polyamine metabolism and organ integrity. Methods: Pancreatic SSAT activity, polyamine pools, pancreatic histology and plasma &#33 -amylase activity were determined after different doses of gossypol. Results: Gossypol increased pancreatic putrescine and decreased spermidine and spermine pools in normal rats accompanied by tissue oedema and significantly elevated plasma amylase activity. In transgenic rats, the drug strikingly induced SSAT, profoundly depleted the higher polyamines and caused distinct pancreatitis. The combination of gossypol at doses harmless to transgenic pancreas with an inhibitor of polyamine oxidase caused massive synergistic induction of SSAT, profound depletion of the polyamine pools and acute pancreatitis. Conclusions: The results indicate that gossypol induces pancreatitis through an activation of polyamine catabolism.     

Gossypol activates pancreatic polyamine catabolism in normal rats and induces acute pancreatitis in transgenic rats over-expressing spermidine/spermine N1-acetyltransferase

BACKGROUND: The male antifertility agent gossypol has been reported to induce spermidine/spermine N1-acetyltransferase (SSAT) in canine prostate cells. As SSAT is the rate-controlling enzyme in the catabolism of the polyamines and is involved in the development of acute pancreatitis in a recent transgenic rat model, we exposed normal and transgenic rats over-expressing SSAT to gossypol to evaluate its effect on pancreatic polyamine metabolism and organ integrity. METHODS: Pancreatic SSAT activity, polyamine pools, pancreatic histology and plasma 2-amylase activity were determined after different doses of gossypol. RESULTS: Gossypol increased pancreatic putrescine and decreased spermidine and spermine pools in normal rats accompanied by tissue oedema and significantly elevated plasma amylase activity. In transgenic rats, the drug strikingly induced SSAT, profoundly depleted the higher polyamines and caused distinct pancreatitis. The combination of gossypol at doses harmless to transgenic pancreas with an inhibitor of polyamine oxidase caused massive synergistic induction of SSAT, profound depletion of the polyamine pools and acute pancreatitis. CONCLUSIONS: The results indicate that gossypol induces pancreatitis through an activation of polyamine catabolism.     

Modulation of Kv channel alpha/beta subunit interactions

Voltage-gated K(+) channels comprise the largest and most diverse class of ion channels. These channels establish the resting membrane potential and modulate the frequency and duration of action potentials in nerve and muscle, as well as being the targets of several antiarrhythmic drugs in the heart. The multiplicity of Kv channel function is further enhanced through modulation by accessory beta subunits, which confer rapid inactivation, alter current amplitudes, and promote cell surface expression. In addition, alpha/beta interactions are also influenced by second messenger pathways. Recent evidence demonstrates that phosphorylation of Kv channel alpha and/or beta subunits may dramatically affect channel properties. The functional response of different K(+) channel subunits to activation of protein kinases represents not only a means to modulate subunit interactions, but also another mechanism for K(+) channel diversity in vivo.     

Activation of polyamine catabolism in transgenic rats induces acute pancreatitis

Polyamines are required for optimal growth and function of cells. Regulation of their cellular homeostasis is therefore tightly controlled. The key regulatory enzyme for polyamine catabolism is the spermidine/spermine N(1)-acetyltransferase (SSAT). Depletion of cellular polyamines has been associated with inhibition of growth and programmed cell death. To investigate the physiological function SSAT, we generated a transgenic rat line overexpressing the SSAT gene under the control of the inducible mouse metallothionein I promoter. Administration of zinc resulted in a marked induction of pancreatic SSAT, overaccumulation of putrescine, and appearance of N(1)-acetylspermidine with extensive depletion of spermidine and spermine in transgenic animals. The activation of pancreatic polyamine catabolism resulted in acute pancreatitis. In nontransgenic animals, an equal dose of zinc did not affect pancreatic polyamine pools, nor did it induce pancreatitis. Acetylated polyamines, products of the SSAT-catalyzed reaction, are metabolized further by the polyamine oxidase (PAO) generating hydrogen peroxide, which might cause or contribute to the pancreatic inflammatory process. Administration of specific PAO inhibitor, MDL72527 [N(1),N(2)-bis(2,3-butadienyl)-1,4-butanediamine], however, did not affect the histological score of the pancreatitis. Induction of SSAT by the polyamine analogue N(1),N(11)-diethylnorspermine reduced pancreatic polyamines levels only moderately and without signs of organ inflammation. In contrast, the combination of N(1), N(11)-diethylnorspermine with MDL72527 dramatically activated SSAT, causing profound depletion of pancreatic polyamines and acute pancreatitis. These results demonstrate that acute induction of SSAT leads to pancreatic inflammation, suggesting that sufficient pools of higher polyamine levels are essential to maintain pancreatic integrity. This inflammatory process is independent of the production of hydrogen peroxide by PAO.     

Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K+ channels

Local anesthetics, commonly used for treating cardiac arrhythmias, pain, and seizures, are best known for their inhibitory effects on voltage-gated Na(+) channels. Cardiovascular and central nervous system toxicity are unwanted side-effects from local anesthetics that cannot be attributed to the inhibition of only Na(+) channels. Here, we report that extracellular application of the membrane-permeant local anesthetic bupivacaine selectively inhibited G protein-gated inwardly rectifying K(+) channels (GIRK:Kir3) but not other families of inwardly rectifying K(+) channels (ROMK:Kir1 and IRK:Kir2). Bupivacaine inhibited GIRK channels within seconds of application, regardless of whether channels were activated through the muscarinic receptor or directly via coexpressed G protein G(beta)gamma subunits. Bupivacaine also inhibited alcohol-induced GIRK currents in the absence of functional pertussis toxin-sensitive G proteins. The mutated GIRK1 and GIRK2 (GIRK1/2) channels containing the high-affinity phosphatidylinositol 4,5-bisphosphate (PIP(2)) domain from IRK1, on the other hand, showed dramatically less inhibition with bupivacaine. Surprisingly, GIRK1/2 channels with high affinity for PIP(2) were inhibited by ethanol, like IRK1 channels. We propose that membrane-permeant local anesthetics inhibit GIRK channels by antagonizing the interaction of PIP(2) with the channel, which is essential for G(beta)gamma and ethanol activation of GIRK channels.     

Structures of wild-type and mutant human spermidine/spermine N1-acetyltransferase, a potential therapeutic drug target

Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels in human cells, as acetylation of spermidine and spermine triggers export or degradation. Increased intracellular polyamine levels accompany several types of cancers as well as other human diseases, and compounds that affect the expression, activity, or stability of SSAT are being explored as potential therapeutic drugs. We have expressed human SSAT from the cloned cDNA in Escherichia coli and have determined high-resolution structures of wild-type and mutant SSAT, as the free dimer and in binary and ternary complexes with CoA, acetyl-CoA (AcCoA), spermine, and the inhibitor N1,N11bis-(ethyl)-norspermine (BE-3-3-3). These structures show details of binding sites for cofactor, substrates, and inhibitor and provide a framework to understand enzymatic activity, mutations, and the action of potential drugs. Two dimer conformations were observed: a symmetric form with two open surface channels capable of binding substrate or cofactor, and an asymmetric form in which only one of the surface channels appears capable of binding and acetylating polyamines. SSAT was found to self-acetylate lysine-26 in the presence of AcCoA and absence of substrate, a reaction apparently catalzyed by AcCoA bound in the second channel of the asymmetric dimer. These unexpected and intriguing complexities seem likely to have some as yet undefined role in regulating SSAT activity or stability as a part of polyamine homeostasis. Sequence signatures group SSAT with proteins that appear to have thialysine Nepsilon-acetyltransferase activity.     

Molecular basis of Kir6.2 mutations associated with neonatal diabetes or neonatal diabetes plus neurological features

Inwardly rectifying potassium channels (Kir channels) control cell membrane K(+) fluxes and electrical signaling in diverse cell types. Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive (K(ATP)) channel, cause permanent neonatal diabetes mellitus (PNDM). For some mutations, PNDM is accompanied by marked developmental delay, muscle weakness, and epilepsy (severe disease). To determine the molecular basis of these different phenotypes, we expressed wild-type or mutant (R201C, Q52R, or V59G) Kir6.2/sulfonylurea receptor 1 channels in Xenopus oocytes. All mutations increased resting whole-cell K(ATP) currents by reducing channel inhibition by ATP, but, in the simulated heterozygous state, mutations causing PNDM alone (R201C) produced smaller K(ATP) currents and less change in ATP sensitivity than mutations associated with severe disease (Q52R and V59G). This finding suggests that increased K(ATP) currents hyperpolarize pancreatic beta cells and impair insulin secretion, whereas larger K(ATP) currents are required to influence extrapancreatic cell function. We found that mutations causing PNDM alone impair ATP sensitivity directly (at the binding site), whereas those associated with severe disease act indirectly by biasing the channel conformation toward the open state. The effect of the mutation on ATP sensitivity in the heterozygous state reflects the different contributions of a single subunit in the Kir6.2 tetramer to ATP inhibition and to the energy of the open state. Our results also show that mutations in the slide helix of Kir6.2 (V59G) influence the channel kinetics, providing evidence that this domain is involved in Kir channel gating, and suggest that the efficacy of sulfonylurea therapy in PNDM may vary with genotype.     

Inflammatory cytokines stimulate vascular smooth muscle cells locomotion and growth by enhancing alpha5beta1 integrin expression and function

The formation of atherosclerotic lesions requires the migration of vascular smooth muscle cells from the media into the intima of the artery and their proliferation. These events, which are preceded and accompanied by inflammation, are modulated by integrin receptors linking vascular smooth muscle cells to extracellular matrix molecules. Among them, fibronectin induces vascular smooth muscle cells to acquire the phenotype they show in the atherosclerotic plaque. Here we show that amounts of interleukin-1 beta, tumor necrosis factor alpha and interferon-gamma as possibly released by activated immune cells infiltrating atherosclerotic lesions, upregulate vascular smooth muscle cell expression of the alpha5beta1 integrin, a fibronectin receptor. This improves vascular smooth muscle cell capability of migrating toward soluble or anchored fibronectin and of adhering to immobilized fibronectin. The latter effect, in turn, augments vascular smooth muscle cell proliferative response to mitogens, as suggested by the increase of intracellular pH. Finally, the effects that inflammatory cytokines have on vascular smooth muscle cell locomotion and growth, are specifically blocked by anti-alpha5beta1 antibodies. As fibronectin and alpha5beta1 levels are augmented in vivo in the atherosclerotic plaques, these findings support the use of integrin antagonists as potential adjuvants in atherosclerosis treatment.     

Interaction of alpha9beta1 integrin with thrombospondin-1 promotes angiogenesis

Thrombospondin-1 is a multifunctional protein interacting with several cell surface receptors including integrins. We found that it is a ligand for alpha9beta1 integrin, and has an integrin binding site within its N-terminal domain (NoC1). Interaction of thrombospondin-1 and its recombinant NoC1 domain with alpha9beta1 integrin was confirmed in ELISA and cell adhesion assays. Binding of NoC1 to cells expressing alpha9beta1 integrin activated signaling proteins such as Erk1/2 and paxillin. Blocking of this integrin by monoclonal antibody and the met-leu-asp-disintegrin inhibited dermal human microvascular endothelial cell proliferation and NoC1-induced migration of these cells. Immunohistochemical studies revealed that alpha9beta1 is expressed on microvascular endothelium in several organs including skin, lung, heart and brain. NoC1 induced neovascularization in an experimental quail chorioallantoic membrane system and Matrigel plug formation assay in mice. This proangiogenic activity of NoC1 in vivo was inhibited by alpha9beta1 inhibitors. In summary, our results revealed that alpha9beta1 integrin expressed on microvascular endothelial cells interacts with thrombospondin-1, and this interaction is involved in modulation of angiogenesis.     

VEGFR-1 (FLT-1), beta1 integrin, and hERG K+ channel for a macromolecular signaling complex in acute myeloid leukemia: role in cell migration and clinical outcome

Leukemia cell motility and transendothelial migration into extramedullary sites are regulated by angiogenic factors and are considered unfavorable prognostic factors in acute leukemias. We have studied cross talk among (1) the vascular endothelial growth factor receptor-1, FLT-1; (2) the human eag-related gene 1 (hERG1) K(+) channels; and (3) integrin receptors in acute myeloid leukemia (AML) cells. FLT-1, hERG1, and the beta(1) integrin were found to form a macromolecular signaling complex. The latter mostly recruited the hERG1B isoform of hERG1 channels, and its assembly was necessary for FLT-1 signaling activation and AML cell migration. Both effects were inhibited when hERG1 channels were specifically blocked. A FLT-1/hERG1/beta(1) complex was also observed in primary AML blasts, obtained from a population of human patients. The co-expression of FLT-1 and hERG1 conferred a pro-migratory phenotype to AML blasts. Such a phenotype was also observed in vivo. The hERG1-positive blasts were more efficient in invading the peripheral circulation and the extramedullary sites after engraftment into immunodeficient mice. Moreover, hERG1 expression in leukemia patients correlated with a higher probability of relapse and shorter survival periods. We conclude that in AML, hERG1 channels mediate the FLT-1-dependent cell migration and invasion, and hence confer a greater malignancy.     

The molecular basis of chloroquine block of the inward rectifier Kir2.1 channel

Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K(+) current I(K1) and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac I(K1). The voltage- and K(+)-dependence of chloroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion-channel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block I(K1) even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties.     

Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy

The ATP-sensitive potassium channel (K(ATP)) regulates insulin secretion in pancreatic beta cells. Loss of functional K(ATP) channels because of mutations in either the SUR1 or Kir6.2 channel subunit causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We investigated the molecular mechanism by which a single phenylalanine deletion in SUR1 (DeltaF1388) causes PHHI. Previous studies have shown that coexpression of DeltaF1388 SUR1 with Kir6.2 results in no channel activity. We demonstrate here that the lack of functional expression is due to failure of the mutant channel to traffic to the cell surface. Trafficking of K(ATP) channels requires that the endoplasmic reticulum-retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during channel assembly. To ask whether DeltaF1388 SUR1 forms functional channels with Kir6.2, we inactivated the RKR signal in DeltaF1388 SUR1 by mutation to AAA (DeltaF1388 SUR1(AAA)). Inactivation of similar endoplasmic reticulum-retention signals in the cystic fibrosis transmembrane conductance regulator has been shown to partially overcome the trafficking defect of a cystic fibrosis transmembrane conductance regulator mutation, DeltaF508. We found that coexpression of DeltaF1388 SUR1(AAA) with Kir6.2 led to partial surface expression of the mutant channel. Moreover, mutant channels were active. Compared with wild-type channels, the mutant channels have reduced ATP sensitivity and do not respond to stimulation by MgADP or diazoxide. The RKR --> AAA mutation alone has no effect on channel properties. Our results establish defective trafficking of K(ATP) channels as a molecular basis of PHHI and show that F1388 in SUR1 is critical for normal trafficking and function of K(ATP) channels.     

A functional role of the C-terminal 42 amino acids of SUR2A and SUR2B in the physiology and pharmacology of cardiovascular ATP-sensitive K(+) channels

The ATP-sensitive K(+) (K(ATP)) channel is composed of four pore-forming Kir6.2 subunits and four sulfonylurea receptors (SUR). Intracellular ATP inhibits K(ATP) channels through Kir6.2. SUR is an ABC protein bearing transmembrane domains and two nucleotide-binding domains (NBD1 and NBD2). SUR increases the open probability of K(ATP) channels by interacting with ATP and ADP through NBDs and with K(+) channel openers such as nicorandil through its transmembrane domain. Because NBDs and the drug receptor allosterically interact with each other, nucleotides and drugs probably activate K(ATP) channels by causing the same conformational change of SUR. SUR2A and SUR2B have the identical drug receptor and NBDs and differ only in the C-terminal 42 amino acids (C42). Nonetheless, nicorandil ~100 times more potently activates SUR2B/Kir6.2 than SUR2A/Kir6.2 channels. Based on our allosteric model, we have analyzed the interaction between NBDs and the drug receptor in SUR2A and SUR2B and found that both nucleotide-bound NBD1 and NBD2 more strongly induce the conformational change in SUR2B than SUR2A. Therefore, C42 modulates the function of not only NBD2 which is close to C42 in a primary structure but NBD1 which is more than 630 amino acid N-terminal to C42. This raises the possibility that in the presence of nucleotides, NBD1 and NBD2 dimerize to induce the conformational change and that the dimerization enables C42 to gain access to both NBDs. Modulation of the nucleotide-NBD1 and -NBD2 interactions by C42 would determine the stability of the nucleotide-dependent dimer and thus, the physiological and pharmacological properties of K(ATP) channels.     

Specificity of activation by phosphoinositides determines lipid regulation of Kir channels

Phosphoinositides are critical regulators of ion channel and transporter activity. Defects in interactions of inwardly rectifying potassium (Kir) channels with phosphoinositides lead to disease. ATP-sensitive K(+) channels (K(ATP)) are unique among Kir channels in that they serve as metabolic sensors, inhibited by ATP while stimulated by long-chain (LC) acyl-CoA. Here we show that K(ATP) are the least specific Kir channels in their activation by phosphoinositides and we demonstrate that LC acyl-CoA activation of these channels depends on their low phosphoinositide specificity. We provide a systematic characterization of phosphoinositide specificity of the entire Kir channel family expressed in Xenopus oocytes and identify molecular determinants of such specificity. We show that mutations in the Kir2.1 channel decreasing phosphoinositide specificity allow activation by LC acyl-CoA. Our data demonstrate that differences in phosphoinositide specificity determine the modulation of Kir channel activity by distinct regulatory lipids.     

Laminin isoform-specific promotion of adhesion and migration of human bone marrow progenitor cells

Laminins are alphabetagamma heterotrimeric extracellular proteins that regulate cellular functions by adhesion to integrin and nonintegrin receptors. Laminins containing alpha4 and alpha5 chains are expressed in bone marrow, but their interactions with hematopoietic progenitors are unknown. We studied human bone marrow cell adhesion to laminin-10/11 (alpha5beta1gamma1/alpha5beta2gamma1), laminin-8 (alpha4beta1gamma1), laminin-1 (alpha1beta1gamma1), and fibronectin. About 35% to 40% of CD34(+) and CD34(+)CD38(-) stem and progenitor cells adhered to laminin-10/11, and 45% to 50% adhered to fibronectin, whereas they adhered less to laminin-8 and laminin-1. Adhesion of CD34(+)CD38(-) cells to laminin-10/11 was maximal without integrin activation, whereas adhesion to other proteins was dependent on protein kinase C activation by 12-tetradecanoyl phorbol-13-acetate (TPA). Fluorescence-activated cell-sorting (FACS) analysis showed expression of integrin alpha6 chain on most CD34(+) and CD34(+)CD38(-) cells. Integrin alpha6 and beta1 chains were involved in binding of both cell fractions to laminin-10/11 and laminin-8. Laminin-10/11 was highly adhesive to lineage-committed myelomonocytic and erythroid progenitor cells and most lymphoid and myeloid cell lines studied, whereas laminin-8 was less adhesive. In functional assays, both laminin-8 and laminin-10/11 facilitated stromal-derived factor-1alpha (SDF-1alpha)-stimulated transmigration of CD34(+) cells, by an integrin alpha6 receptor-mediated mechanism. In conclusion, we demonstrate laminin isoform-specific adhesive interactions with human bone marrow stem, progenitor, and more differentiated cells. The cell-adhesive laminins affected migration of hematopoietic progenitors, suggesting a physiologic role for laminins during hematopoiesis.     

Shear stress regulates the endothelial Kir2.1 ion channel

Endothelial cells (ECs) line the mammalian vascular system and respond to the hemodynamic stimulus of fluid shear stress, the frictional force produced by blood flow. When ECs are exposed to shear stress, one of the fastest responses is an increase of K(+) conductance, which suggests that ion channels are involved in the early shear stress response. Here we show that an applied shear stress induces a K(+) ion current in cells expressing the endothelial Kir2.1 channel. This ion current shares the properties of the shear-induced current found in ECs. In addition, the shear current induction can be specifically prevented by tyrosine kinase inhibition. Our findings identify the Kir2.1 channel as an early component of the endothelial shear response mechanism.     

MMP-9 in B-cell chronic lymphocytic leukemia is up-regulated by alpha4beta1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration

B-cell chronic lymphocytic leukemia (B-CLL) progression is determined by malignant cell extravasation and lymphoid tissue infiltration. We have studied the role and regulation of matrix metalloproteinase-9 (MMP-9) in B-CLL cell migration and invasion. Adhesion of B-CLL cells to the fibronectin fragment FN-H89, VCAM-1, or TNF-alpha-activated human umbilical vein endothelial cells (HUVECs) up-regulated MMP-9 production, measured by gelatin zymography. This effect was mediated by alpha4beta1 integrin and required PI3-K/Akt signaling. The chemokine CXCL12 also up-regulated MMP-9, independently of alpha4beta1 and involving ERK1/2 but not Akt activity. Accordingly, alpha4beta1 engagement activated the PI3-K/Akt/NF-kappaB pathway, while CXCL12/CXCR4 interaction activated ERK1/2/c-Fos signaling. Anti-MMP-9 antibodies, the MMP-9 inhibitor TIMP-1, or transfection with 3 different MMP-9 siRNAs significantly blocked migration through Matrigel or HUVECs. Cell-associated MMP-9 was mainly at the membrane and contained the proactive and mature forms. Moreover, B-CLL cells formed podosomes upon adhesion to FN-H89, VCAM-1, or fibronectin; MMP-9 localized to podosomes in a PI3-K-dependent manner and degraded a fibronectin/gelatin matrix. Our results are the first to show that MMP-9 is physiologically regulated by alpha4beta1 integrin and CXCL12 and plays a key role in cell invasion and transendothelial migration, thus contributing to B-CLL progression. MMP-9 could therefore constitute a target for treatment of this malignancy.     

Engineered specific and high-affinity inhibitor for a subtype of inward-rectifier K+ channels

Inward-rectifier K(+) (Kir) channels play many important biological roles and are emerging as important therapeutic targets. Subtype-specific inhibitors would be useful tools for studying the channels' physiological functions. Unfortunately, available K(+) channel inhibitors generally lack the necessary specificity for their reliable use as pharmacological tools to dissect the various kinds of K(+) channel currents in situ. The highly conserved nature of the inhibitor targets accounts for the great difficulty in finding inhibitors specific for a given class of K(+) channels or, worse, individual subtypes within a class. Here, by modifying a toxin from the honey bee venom, we have successfully engineered an inhibitor that blocks Kir1 with high (1 nM) affinity and high (>250-fold) selectivity over many commonly studied Kir subtypes. This success not only yields a highly desirable tool but, perhaps more importantly, demonstrates the practical feasibility of engineering subtype-specific K(+) channel inhibitors.     

Focus on Kir6.2: a key component of the ATP-sensitive potassium channel

ATP-sensitive potassium (K(ATP)) channels are found in a wide variety of cell types where they couple cell metabolism to electrical activity. In glucose-sensing tissues, these channels respond to fluctuating changes in blood glucose concentration, but in other tissues they are activated only under ischemic conditions or in response to hormonal stimulation. Although K(ATP) channels in different tissues have different regulatory subunits, in almost all cases (except vascular smooth muscle) the pore-forming subunit is the inwardly rectifying K(+) channel Kir6.2. This article reviews recent studies of Kir6.2, focussing on the relation between channel structure and function, and on naturally occurring mutations in Kir6.2 that lead to human disease. New insights into the location of the ATP-binding site, the permeation pathway for K(+), and the gating of the pore provided by homology modelling are discussed in relation to functional studies. Gain-of-function mutations in Kir6.2 cause permanent neonatal diabetes mellitus (PNDM) by reducing the ATP sensitivity of the K(ATP) channel and increasing the K(ATP) current, which is predicted to inhibit beta-cell electrical activity and insulin secretion. Mutations at specific residues, that cause a greater decrease in ATP sensitivity, are associated with additional neurological symptoms. The molecular mechanism underlying the differences in ATP sensitivity produced by these two classes of mutations is discussed. We speculate on how some mutations lead to neurological disease and why no obvious cardiac symptoms are observed. We also consider the implications of these studies for type-2 diabetes.