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ABSTRACT: ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.
Journal of Membrane Biology 12/2005; 208(1):65-76. · 1.81 Impact Factor
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[show abstract]
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ABSTRACT: ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent
crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study
we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting
the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion
L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of
scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence
of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms
of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that
the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity
of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.
Journal of Membrane Biology 10/2005; 208(1):65-76. · 1.81 Impact Factor
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ABSTRACT: Blockade of the CFTR chloride channel by glibenclamide was studied in Xenopus oocytes using two-electrode voltage-clamp recordings, macropatch recordings, and summations of single-channel currents, in order to test a kinetic model recently developed by us from single-channel experiments. Both the forward and reverse macroscopic reactions, at negative and positive membrane potential V(M), respectively, were slow in comparison to those reactions for other CFTR pore blockers such as DPC and NPPB, resulting in prominent relaxations on the order of tens of milliseconds. The rate of the reverse reaction was voltage-dependent, and dependent on the Cl(-) driving force, while that of the forward reaction was not. In inside-out macropatches, block and relief from block occurred in two distinct phases that differed in apparent affinity. The results are consistent with the presence of multiple glibenclamide binding sites in CFTR, with varying affinity and voltage dependence; they support the kinetic model and suggest experimental approaches for identification of those sites by mutagenesis.
Journal of Membrane Biology 11/2004; 201(3):139-55. · 1.81 Impact Factor
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ABSTRACT: The objective of the present study was to clarify the mechanism by which the sulfonylurea drug, glibenclamide, inhibits single CFTR channels in excised patches from Xenopus oocytes. Glibenclamide blocks the open pore of the channel via binding at multiple sites with varying kinetics. In the absence of glibenclamide, open-channel bursts exhibited a flickery intraburst closed state (C1); this is due to block of the pore by the pH buffer, TES. Application of 25 microM glibenclamide to the cytoplasmic solution resulted in the appearance of two drug-induced intraburst closed states (C2, C3) of widely different duration, which differed in pH-dependence. The kinetics of interaction with the C3 state, but not the C2 state, were strongly voltage-dependent. The durations of both the C2 and C3 states were concentration-dependent, indicating a non-linear reaction scheme. Application of drug also increased the burst duration, which is consistent with an open-channel blocking mechanism. A kinetic model is proposed. These results indicate that glibenclamide interacts with open CFTR channels in a complex manner, involving interactions with multiple binding sites in the channel pore.
Journal of Membrane Biology 06/2004; 199(1):15-28. · 1.81 Impact Factor
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ABSTRACT: The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl(-) channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl(-) and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl(-) and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl(-) and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.
AJP Lung Cellular and Molecular Physiology 11/2001; 281(4):L852-67. · 3.66 Impact Factor
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ABSTRACT: The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.
The Journal of General Physiology 11/2001; 118(4):407-31. · 3.84 Impact Factor
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ABSTRACT: The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel with distinctive kinetics. At the whole-cell level, CFTR currents in response to voltage steps are time independent for wild type and for the many mutants reported so far. Single channels open for periods lasting up to tens of seconds; the openings are interrupted by brief closures at hyperpolarized, but not depolarized, potentials. Here we report a serine-to-phenylalanine mutation (S1118F) in the 11th transmembrane domain that confers voltage-dependent, single-exponential current relaxations and moderate inward rectification of the macroscopic currents upon expression in Xenopus oocytes. At steady state, the S1118F-CFTR single-channel conductance rectifies, corresponding to the whole-cell rectification. In addition, the open-channel burst duration is decreased 10-fold compared with wild-type channels. S1118F-CFTR currents are blocked in a voltage-dependent manner by diphenylamine-2-carboxylate (DPC); the affinity of S1118F-CFTR for DPC is similar to that of the wild-type channel, but blockade exhibits moderately reduced voltage dependence. Selectivity of the channel to a range of anions is also affected by this mutation. Furthermore, the permeation properties change during the relaxations, which suggests that there is an interaction between gating and permeation in this mutant. The existence of a mutation that confers voltage dependence upon CFTR currents and that changes kinetics and permeation properties of the channel suggests a functional role for the 11th transmembrane domain in the pore in the wild-type channel.
Biophysical Journal 08/2000; 79(1):298-313. · 3.65 Impact Factor
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ABSTRACT: Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at -100 mV. The affinity for NPPB calculated from single-channel block, K(D) = 35 microm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type (WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB. We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects (i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes for overlapping regions in the pore.
Journal of Membrane Biology 06/2000; 175(1):35-52. · 1.81 Impact Factor
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ABSTRACT: In A6 distal nephron cells, short-circuit current (Isc) was increased by basolateral exposure to prostaglandin E2 (PGE2; peak response at 1 microM). The effect was only partially abolished by either apical amiloride, an Na+ channel blocker, or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a Cl- channel blocker. In apical cell-attached patches, we observed a 7-pS Cl- channel with a linear current-voltage relationship, a reversal potential near resting membrane potential, and open probability > 0.5. The channel was blocked by diphenylamine-2-carboxylate, glibenclamide, and NPPB but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The frequency of observed Cl- channel activity increased 7-fold with 10-min exposure to PGE2 and 3.7-fold with longer (10-50 min) exposure to PGE2. The PGE2-induced increase in Cl- channel activity was due primarily to an increase in the number of functional channels. The following conclusions were made: 1) activation of apical, 7-pS Cl- channels in A6 cells accounts for the PGE2-induced increase in the amiloride-insensitive Isc, and 2) 7-pS Cl- channel activation was mediated via an increase in channel density without substantial effects on channel kinetics.
The American journal of physiology 09/1997; 273(2 Pt 1):C548-57.
