1,721,201 research outputs found
Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains
No full textThe use of substances that could activate the defective chloride channels of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) has been suggested as possible therapy for cystic fibrosis. Using epithelia formed by cells stably transfected with wildtype or mutant (G551D, G1349D) CFTR, we estimated the apparent dissociation constant, K(D), of a series of CFTR activators by measuring the increase in the apical membrane current. Modification of apparent K(D) of CFTR activators by mutations of the nucleotide-binding domains (NBDs) suggests that the binding site might be in these regions. The human NBD structure was predicted by homology with murine NBD1. An NBD1-NBD2 complex was constructed by overlying monomers to a bacterial ABC transporter NBD dimer in the "head-to-tail" conformation. Binding sites for CFTR activators were predicted by molecular docking. Comparison of theoretical binding free energy estimated in the model to free energy estimated from the apparent dissociation constants, K(D), resulted in a remarkably good correlation coefficient for one of the putative binding sites, located in the interface between NBD1 and NBD2
High conductance pathways in mitochondrial membranes
No full textThe outer and inner membranes of mitochondria have recently been studied with the patch clamp technique. What has emerged is still an ill-defined picture for either membrane, primarily for the wide range of conductances found. Interestingly, however, a few conductances (in the range of 10-80 pS) seem to be ubiquitously distributed. Parallel studies in situ and in reconstituted systems have allowed the assignment to distinct membrane locations of some conductances, whose physiological role is, however, not yet elucidated
Channels in mitochondrial membranes: knowns, unknowns and prospects for the future
No full textRapid diffusion of hydrophilic molecules across the outer membrane of mitochondria has been related to the presence of a protein of 29 to 37 kDa, called voltage-dependent anion channel (VDAC), able to generate large aqueous pores when integrated in planar lipid bilayers. Functional properties of VDAC from different origins appear highly conserved in artificial membranes: at low transmembrane potentials, the channel is in a highly conducting state, but a raise of the potential (both positive and negative) reduces drastically the current and changes the ionic selectivity from slightly anionic to cationic. It has thus been suggested that VDAC is not a mere molecular sieve but that it may control mitochondrial physiology by restricting the access of metabolites of different valence in response to voltage and/or by interacting with a soluble protein of the intermembrane space. The latest application of the patch clamp and tip-dip techniques, however, has indicated both a different electric behavior of the outer membrane and that other proteins may play a role in the permeation of molecules. Biochemical studies, use of site-directed mutants, and electron microscopy of two-dimensional crystal arrays of VDAC have contributed to propose a monomeric beta barrel as the structural model of the channel. An important insight into the physiology of the inner membrane of mammalian mitochondria has come from the direct observation of the membrane with the patch clamp. A slightly anionic, voltage-dependent conductance of 107 pS and one of 9.7 pS, K(+)-selective and ATP-sensitive, are the best characterized at the single channel level. Under certain conditions, however, the inner membrane can also show unselective nS peak transitions, possibly arising from a cooperative assembly of multiple substrates
Characterization of a murine gene homologous to the bovine CaCC chloride channel
No full textThe bovine CaCC protein is a putative Ca2+-dependent Cl- channel of airway epithelial cells. Therefore, CaCC proteins could contribute to transepithelial Cl- transport and accordingly modify the phenotype of cystic fibrosis (CF) patients. We have identified a murine EST containing a full-length cDNA coding for a 902-amino-acid protein highly homologous to bovine CaCC. The murine gene (mCaCC) maps to chromosome 3 at the H2-H3 band and is expressed, as indicated by Northern blot analysis, in mouse skin and kidney but not in brain, heart, lung or testis. RT-PCR indicates a low expression in tracheal epithelial cells. Heterologous expression of mCaCC in Xenopus oocytes elicits membrane currents that are anion-selective and inhibited by DIDS and by niflumic acid, a blocker of the endogenous chloride current in oocytes. The identification of genes belonging to the CaCC family will help to evaluate their role as ion channels or channel regulators and their actual contribution to epithelial chloride transport
Attivatori del trasporto ionico della CFTR: identificazione e modellazione molecolare dei siti leganti
No full textAccumulation of long-lasting inactivation in rat brain K+-channels
No full textWe studied the phenomenon of cumulative inactivation in the voltage-dependent K+ channels of the Shaker-related subfamily Kv1 cloned from rat brain and expressed in Xenopus oocytes. In Kv1.4, repetitive stimulations at intervals shorter than 20 s produce cumulative inactivation even for brief stimuli that elicit K+ currents which do not show any significant decline during the depolarising pulse. These effects are absent or greatly reduced in the clones Kv1.1, Kv1.3, Kv1.5 and Kv1.6, and in the deletion mutant Kv1.4-delta-110, characterised by lack of "fast" (N-type) inactivation. We find that the inactivation caused by a single pulse increases after the pulse while the channels deactivate, and subsides with two time constants, indicating the existence of (at least) two inactivated states: IS, with a slow recovery kinetics and IF, with faster kinetics. In the simplest kinetic scheme accounting for our observations, IF is coupled sequentially to the open state O, while IS can be reached at a fast rate both from IF and from a pre-open, activated state, A, that is in fast equilibrium with O. The accumulation of long-lasting inactivation during the repolarisation is favoured by the prolongation of the lifetime of activated states due to the presence of IF. This explains the smaller accumulation effect observed in channels lacking fast inactivation. The physiological implications of these findings suggest how different channels of the Kv1 subfamily can affect differently the firing behaviour of neurones
Changes of N-Methyl-D-Aspartate activated channels of cerebellar granule cells with days in culture
No full textACTIVATION AND DEACTIVATION PROPERTIES OF RAT-BRAIN K+ CHANNELS OF THE SHAKER-RELATED SUBFAMILY
No full textWe studied the activation properties of members of the Shaker-related subfamily of voltage-gated K+ channels cloned from rat brain and expressed in Xenopus oocytes. We find that Kv1.1, Kv1.4, Kv1.5, and Kv1.6 have similar activation and deactivation kinetics. The k+ currents produced by step depolarisations increase with a sigmoidal time course that can be described by a delay and by the derivative of the current at the inflection point. The delay tends to zero and the logarithmic derivative seems to approach a finite value at large positive voltages, but these asymptotic values are not yet reached at +80 mV. Deactivation of the currents upon stepping to negative membrane potentials below -60 mV is fairly well described by a single exponential. The decrease of the deactivation time constant at increasingly negative voltages tends to become less steep, indicating that this parameter also has a finite limiting value, which is not yet reached, however, at -160 mV. The various clones studied have very similar voltage dependencies of activation with half-activation voltages ranging between -50 and -11 mV and maximum steepness yielding and e-fold change for voltage increments between 3.8 and 7.0 mV. The shallower activation curve of Kv1.4 is likely to be due to coupling with the fast inactivation process present in this clone
Biophysical characteristics of swelling-activated Cl- channels in human tracheal 9HTEo-cells
No full textThe question of whether a single molecule can account for every observed swelling-activated Cl- current deserves to be addressed and biophysical description seems to be an adequate criterion to classify these channels. We studied the biophysical properties of swelling-activated Cl- currents in 9HTEo-cells using whole-cell and outside-out patch clamp recordings. Hypotonic shock activated outwardly rectifying currents that inactivated at potentials higher than 20 mV. The decay phase of the current was well fitted by two exponential functions and both time constants were voltage-dependent. Two voltage-dependent time constants were also necessary to describe reactivation. The midpoint of current inactivation was 54 mV. The voltage dependence of kinetics did not significantly change by modifying the extracellular NaCl concentration while the inactivation midpoint slightly shifted. In conclusion, our results indicate that the voltage-dependent properties of the swelling-activated Cl- currents in 9HTEo- cells are largely independent from the extracellular ionic strength and the extracellular Cl- concentration. Excised patches from cells exposed to hypotonic shock showed single channel currents that inactivated at positive membrane potentials and displayed chord conductance of approximately 60 pS at 100 mV and of approximately 20 pS at -80 mV. The permeability sequence for the single channel was I- > Br- > Cl- > gluconate and currents were blocked by Reactive blue 2. These properties indicate that intermediate conductance outwardly rectifying channels are responsible for the macroscopic swelling-activated current
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