49 research outputs found
Mutation-induced Blocker Permeability and Multiion Block of the CFTR Chloride Channel Pore
Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Clâ channel is blocked by a broad range of anions that bind tightly within the pore. Here we show that the divalent anion Pt(NO2)42â acts as an impermeant voltage-dependent blocker of the CFTR pore when added to the intracellular face of excised membrane patches. Block was of modest affinity (apparent Kd 556 ÎŒM), kinetically fast, and weakened by extracellular Clâ ions. A mutation in the pore region that alters anion selectivity, F337A, but not another mutation at the same site that has no effect on selectivity (F337Y), had a complex effect on channel block by intracellular Pt(NO2)42â ions. Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Current in the presence of Pt(NO2)42â increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO2)42â ions to the extracellular solution. This âpunchthroughâ was prevented by extracellular Clâ ions, reminiscent of a âlock-inâ effect. Relief of block in F337A by Pt(NO2)42â permeation was only observed for blocker concentrations above 300 ÎŒM; as a result, block at very negative voltages showed an anomalous concentration dependence, with an increase in blocker concentration causing a significant weakening of block and an increase in Clâ current. We interpret this effect as reflecting concentration-dependent permeability of Pt(NO2)42â in F337A, an apparent manifestation of an anomalous mole fraction effect. We suggest that the F337A mutation allows intracellular Pt(NO2)42â to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO2)42â ions within the pore promotes their permeation to the extracellular solution
Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore
Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Clâ channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84âT94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore
Role of the Juxtamembrane Region of Cytoplasmic Loop 3 in the Gating and Conductance of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel
Finishing the euchromatic sequence of the human genome
The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers âŒ99% of the euchromatic genome and is accurate to an error rate of âŒ1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead
NM23 proteins: innocent bystanders or local energy boosters for CFTR?
NM23 proteins NDPK-A and -B bind to the cystic fibrosis (CF) protein CFTR in different ways from kinases such as PKA, CK2 and AMPK or linkers to cell calcium such as calmodulin and annexins. NDPK-A (not -B) interacts with CFTR through reciprocal AMPK binding/control, whereas NDPK-B (not -A) binds directly to CFTR. NDPK-B can activate G proteins without ligand-receptor coupling, so perhaps NDPK-B's binding influences energy supply local to a nucleotide-binding site (NBD1) needed for CFTR to function. Curiously, CFTR (ABC-C7) is a member of the ATP-binding cassette (ABC) protein family that does not obey 'clan rules'; CFTR channels anions and is not a pump, regulates disparate processes, is itself regulated by multiple means and is so pleiotropic that it acts as a hub that orchestrates calcium signaling through its consorts such as calmodulin/annexins. Furthermore, its multiple partners make CFTR dance to different tunes in different cellular and subcellular locations as it recycles from the plasma membrane to endosomes. CFTR function in airway apical membranes is inhibited by smoking which has been dubbed 'acquired CF'. CFTR alone among family members possesses a trap for other proteins that it unfurls as a 'fish-net' and which bears consensus phosphorylation sites for many protein kinases, with PKA being the most canonical. Recently, the site of CFTR's commonest mutation has been proposed as a knock-in mutant that alters allosteric control of kinase CK2 by log orders of activity towards calmodulin and other substrates after CFTR fragmentation. This link from CK2 to calmodulin that binds the R region invokes molecular paths that control lumen formation, which is incomplete in the tracheas of some CF-affected babies. Thus, we are poised to understand the many roles of NDPK-A and -B in CFTR function and, especially lumen formation, which is defective in the gut and lungs of many CF babies
Voltage gated potassium channels of cultured rat central neurons.
Whole cell and single channel patch clamp recording were used to study the properties of two types of voltage gated potassium channels in rat neurons. Neurons were dissociated from either the locus coeruleus or hippocampus of neonatal rats and grown in primary cell culture for 6 to 17 days before recording. Cultured neurons from both these brain areas were found to express whole cell A-currents and delayed rectifier currents, similar to those seen in other neurons. The conductance of single A-current channels in locus coeruleus neurons was 14.8pS, although this declined at positive membrane potentials. This reduction in unitary amplitude was shown to be due to voltage dependent block of the channel by intracellular magnesium and sodium ions. The Kd values of the blocking reactions at 0mV membrane potential were 15.7mM for magnesium and 76.0mM for sodium, the Kd of each block decreasing with increasing membrane depolarisation. The conductance of single delayed rectifier channels in hippocampal neurons was 18.4pS. Gating of this channel could be explained by a model in which the channel had one open state, four closed states and three inactivated states. This scheme was not consistent with a Hodgkin-Huxley model of voltage dependent gating, but instead favoured a model whereby gating occurred in a cooperative manner. Gating could be altered by changing the permeant ion from potassium to the less permeable rubidium; this had the effect of specifically slowing all channel closing rate constants. The rubidium permeability of this channel was 75% of the potassium permeability. External tetraethyl ammonium ions blocked delayed rectifier channels at two independent sites, each block having different kinetic properties. The Kd of the kinetically faster block was 53.4?M, whilst that of the slower block was estimated to be between 400 and 800?M
Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating
AbstractOpening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single âgateâ within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)2â ions was reduced when ATP concentration was reduced from 1mM to 10ÎŒM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)2â was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids
Multiple inhibitory effects of Au(CN)2â ions on cystic fibrosis transmembrane conductance regulator Clâ channel currents
Lyotropic pseudohalide anions are potentially useful as high affinity probes of Clâ channel pores. However, the interaction between these pseudohalides and the cystic fibrosis transmembrane conductance regulator (CFTR) Clâ channel have not been described in detail. Here we show that Au(CN)2â ions applied to the intracellular face of membrane patches from stably transfected baby hamster kidney cells inhibit CFTR channel currents by at least two mechanisms, which can be distinguished at the single channel level or by inhibiting channel closure using 2 mm pyrophosphate. Low concentrations (< 10 ÎŒm) of Au(CN)2â significantly reduced CFTR channel open probability. This effect was apparently voltage insensitive, independent of extracellular Clâ concentration, and lost following exposure to pyrophosphate. Higher concentrations of intracellular Au(CN)2â caused an apparent reduction in unitary current amplitude, presumably due to a kinetically fast blocking reaction. This effect, isolated following exposure to pyrophosphate, was strongly voltage dependent (apparent Kd 61.6 ÎŒm at â100 mV and 913 ÎŒm at +60 mV). Both the affinity and voltage dependence of block were highly sensitive to extracellular Clâ concentration. We propose that Au(CN)2â has at least two inhibitory effects on CFTR currents: a high affinity effect on channel gating due to action on a cytoplasmically accessible aspect of the channel and a lower affinity block within the open channel pore. These results offer important caveats for the use of lyotropic pseudohalide anions such as Au(CN)2â as specific high affinity probes of Clâ channel pores