Alternative sulfonylurea receptor expression defines metabolic sensitivity of K-ATP channels in dopaminergic midbrain neurons.
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ATP-sensitive potassium (K-ATP) channels couple the metabolic state to cellular excitability in various tissues. Several isoforms of the K-ATP channel subunits, the sulfonylurea receptor (SUR) and inwardly rectifying K channel (Kir6.X), have been cloned, but the molecular composition and functional diversity of native neuronal K-ATP channels remain unresolved. We combined functional analysis of K-ATP channels with expression profiling of K-ATP subunits at the level of single substantia nigra (SN) neurons in mouse brain slices using an RT-multiplex PCR protocol. In contrast to GABAergic neurons, single dopaminergic SN neurons displayed alternative co-expression of either SUR1, SUR2B or both SUR isoforms with Kir6.2. Dopaminergic SN neurons expressed alternative K-ATP channel species distinguished by significant differences in sulfonylurea affinity and metabolic sensitivity. In single dopaminergic SN neurons, co-expression of SUR1 + Kir6.2, but not of SUR2B + Kir6.2, correlated with functional K-ATP channels highly sensitive to metabolic inhibition. In contrast to wild-type, surviving dopaminergic SN neurons of homozygous weaver mouse exclusively expressed SUR1 + Kir6.2 during the active period of dopaminergic neurodegeneration. Therefore, alternative expression of K-ATP channel subunits defines the differential response to metabolic stress and constitutes a novel candidate mechanism for the differential vulnerability of dopaminergic neurons in response to respiratory chain dysfunction in Parkinson's disease. (+info)
Inward rectification in KATP channels: a pH switch in the pore.
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Inward-rectifier potassium channels (Kir channels) stabilize the resting membrane potential and set a threshold for excitation in many types of cell. This function arises from voltage-dependent rectification of these channels due to blockage by intracellular polyamines. In all Kir channels studied to date, the voltage-dependence of rectification is either strong or weak. Here we show that in cardiac as well as in cloned KATP channels (Kir6.2 + sulfonylurea receptor) polyamine-mediated rectification is not fixed but changes with intracellular pH in the physiological range: inward-rectification is prominent at basic pH, while at acidic pH rectification is very weak. The pH-dependence of polyamine block is specific for KATP as shown in experiments with other Kir channels. Systematic mutagenesis revealed a titratable C-terminal histidine residue (H216) in Kir6.2 to be the structural determinant, and electrostatic interaction between this residue and polyamines was shown to be the molecular mechanism underlying pH-dependent rectification. This pH-dependent block of KATP channels may represent a novel and direct link between excitation and intracellular pH. (+info)
Overexpression of the multidrug resistance-associated protein (MRP1) in human heavy metal-selected tumor cells.
(3/7249)
Cellular and molecular mechanisms involved in the resistance to cytotoxic heavy metals remain largely to be characterized in mammalian cells. To this end, we have analyzed a metal-resistant variant of the human lung cancer GLC4 cell line that we have selected by a step-wise procedure in potassium antimony tartrate. Antimony-selected cells, termed GLC4/Sb30 cells, poorly accumulated antimony through an enhanced cellular efflux of metal, thus suggesting up-regulation of a membrane export system in these cells. Indeed, GLC4/Sb30 cells were found to display a functional overexpression of the multidrug resistance-associated protein MRP1, a drug export pump, as demonstrated by Western blotting, reverse transcriptase-polymerase chain reaction and calcein accumulation assays. Moreover, MK571, a potent inhibitor of MRP1 activity, was found to markedly down-modulate resistance of GLC4/Sb30 cells to antimony and to decrease cellular export of the metal. Taken together, our data support the conclusion that overexpression of functional MRP1 likely represents one major mechanism by which human cells can escape the cytotoxic effects of heavy metals. (+info)
Neural modulation of cephalexin intestinal absorption through the di- and tripeptide brush border transporter of rat jejunum in vivo.
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Intestinal absorption of beta-lactamine antibiotics (e.g., cefixime and cephalexin) has been shown to proceed through the dipeptide carrier system. In a previous study, nifedipine (NFP), an L-type calcium channel blocker, enhanced the absorption of cefixime in vivo but not in vitro, and it was suggested that neural mechanisms might be involved in the effect of NFP. The aim of the present study was to assess the involvement of the nervous system on the intestinal absorption of cephalexin (CFX). To investigate this, we used a single-pass jejunal perfusion technique in rats. NFP and diltiazem enhanced approximately 2-fold the plasma levels of CFX in treated rats versus untreated controls. NFP also increased approximately 2-fold the CFX level in portal plasma and increased urinary excretion of CFX, thus indicating that CFX did effectively increase CFX intestinal absorption. Perfusing high concentrations of dipeptides in the jejunal lumen competitively reduced CFX absorption and inhibited the enhancement of CFX absorption produced by NFP. Hexamethonium and lidocaine inhibited the effect of NFP, whereas atropine, capsaicin, clonidine, and isoproterenol enhanced CFX absorption by the same order of magnitude as NFP. Thus, complex neural networks can modulate the function of the intestinal di- and tripeptide transporter. Sympathetic noradrenergic fibers, intestinal sensory neurons, and nicotinic synapses are involved in the increase of CFX absorption produced by NFP. (+info)
Expression of atrC - encoding a novel member of the ATP binding cassette transporter family in Aspergillus nidulans - is sensitive to cycloheximide.
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A new member of the ABC superfamily of transmembrane proteins in Aspergillus nidulans has been cloned and characterized. The topology of conserved motifs subgroups AtrC in the P-glycoprotein cluster of ABC permeases, the members of this subfamily, are known to participate in multidrug resistance (MDR) in diverse organisms. Alignment results display significant amino acid similarity to AfuMDR1 and AflMDR1 from Aspergillus fumigatus and flavus, respectively. Northern analysis reveals that atrC mRNA levels are 10-fold increased in response to cycloheximide. Evidence for the existence of eight additional hitherto unpublished ABC transporter proteins in A. nidulans is provided. (+info)
MalK forms a dimer independent of its assembly into the MalFGK2 ATP-binding cassette transporter of Escherichia coli.
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The maltose transport complex (MTC) is a member of the ATP-binding cassette superfamily of membrane transport proteins and is a model for understanding the folding and assembly of hetero-oligomeric membrane protein complexes. The MTC is made up of two integral membrane proteins, MalF and MalG, and a peripheral membrane protein, MalK. These proteins associate with a stoichiometry of 1:1:2 to form the complex MalFGK2. In our studies of the oligomerization of this complex, we have shown that the ATP-binding component, MalK, forms a dimer in the absence of MalF and MalG. Epitope-tagged MalK coimmunoprecipitated with wild-type MalK, indicating that the MalK protein forms an oligomer. The relative amounts of tagged and wild-type MalK that were present in the whole cell extracts and in the immunoprecipitated complexes show that the MalK oligomer is a dimer. These hetero-oligomers can also be formed in vitro by mixing two extracts, each containing either tagged or wild-type MalK. The dimerization of MalK was also demonstrated in vivo using the bacteriophage lambda repressor fusion assay. The formation of a MalK dimer in the absence of MalF and MalG may represent an initial step in the assembly pathway of the MTC. (+info)
Glucose-receptive neurones in the rat ventromedial hypothalamus express KATP channels composed of Kir6.1 and SUR1 subunits.
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1. Patch-clamp recordings were made from rat ventromedial hypothalamic neurones in slices of brain tissue in vitro. In cell-attached recordings, removal of extracellular glucose or metabolic inhibition with sodium azide reduced the firing rate of a subpopulation of cells through the activation of a 65 pS channel that was blocked by the sulphonylureas tolbutamide and glibenclamide. 2. In whole-cell patch-clamp recordings, in the absence of ATP in the electrode solution, glucose-receptive neurones gradually hyperpolarized due to the induction of an outward current at -60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide. 3. In recordings where the electrode solution contained 4 mM ATP, this outward current was not observed. Under these conditions, 500 microM diazoxide was found to induce an outward current that was blocked by tolbutamide. 4. In cell-attached recordings diazoxide and the active fragment of leptin (leptin 22-56) reduced the firing rate of glucose-receptive neurones by the activation of a channel with similar properties to that induced by removal of extracellular glucose. 5. Reverse transcription followed by the polymerase chain reaction using cytoplasm from single glucose-receptive neurones demonstrated the expression of the ATP-sensitive potassium (KATP) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2. 6. It is concluded that glucose-receptive neurones within the rat ventromedial hypothalamus exhibit a KATP channel current with pharmacological and molecular properties similar to those reported in other tissues. (+info)
Functional analysis of the promoter of the yeast SNQ2 gene encoding a multidrug resistance transporter that confers the resistance to 4-nitroquinoline N-oxide.
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The yeast gene SNQ2, which encodes a multidrug resistance ABC superfamily protein, is required for resistance to the mutagen 4-nitroquinoline N-oxide (4-NQO). The expression of the SNQ2 gene is under the control of a regulatory network that involves the transcription factor Yrr1p, as well as Pdr1p/Pdr3p (Cui et al., Mol. Microbiol., 29, 1307-1315 (1998)). By 5'-deletion analysis of the promoter by using SNQ2-lacZ fusion constructs, four regions: -745 to -639 (region I), -639 to -578 (region II), -548 to -533 (region III) and -533 to -485 (region IV) were found to be important for SNQ2 expression. Genetic analysis suggested that the site in region IV was responsible for the Yrr1p-mediated SNQ2 expression. A consensus motif known for the binding of Pdr1p/Pdr3p (PDRE) was not found in region IV. (+info)