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L Csanády

Publications and source records attributed to L Csanády.

4 recordsLinked to original sources
PubMed2000

Rapid kinetic analysis of multichannel records by a simultaneous fit to all dwell-time histograms.

A method is presented for rapidly extracting single-channel transition rate constants from patch-clamp recordings containing signals from several channels. The procedure is based on a simultaneous fit of the observed dwell-time distributions for all conductance levels, using a maximum likelihood approach. This algorithm allows estimation of single-channel rate constants in cases where more advanced methods may be impractical because of their extremely long computational time. A correction is included for the limited time resolution of the recording system, according to theory developed by Roux and Sauvé (Biophys. J. 48:149-158, 1985), by accounting for the impact of undetected transitions on the dwell-time distributions, and by introducing an improved practical implementation of a fixed dead time for the case of more than one channel. This feature allows application of the method to noisy data, after filtering. A computer program implementing the method is tested successfully on a variety of simulated multichannel current traces.

Algorithms↗
PubMed2000

Severed molecules functionally define the boundaries of the cystic fibrosis transmembrane conductance regulator's NH(2)-terminal nucleotide binding domain.

The cystic fibrosis transmembrane conductance regulator is a Cl(-) channel that belongs to the family of ATP-binding cassette proteins. The CFTR polypeptide comprises two transmembrane domains, two nucleotide binding domains (NBD1 and NBD2), and a regulatory (R) domain. Gating of the channel is controlled by kinase-mediated phosphorylation of the R domain and by ATP binding, and, likely, hydrolysis at the NBDs. Exon 13 of the CFTR gene encodes amino acids (aa's) 590-830, which were originally ascribed to the R domain. In this study, CFTR channels were severed near likely NH(2)- or COOH-terminal boundaries of NBD1. CFTR channel activity, assayed using two-microelectrode voltage clamp and excised patch recordings, provided a sensitive measure of successful assembly of each pair of channel segments as the sever point was systematically shifted along the primary sequence. Substantial channel activity was taken as an indication that NBD1 was functionally intact. This approach revealed that the COOH terminus of NBD1 extends beyond aa 590 and lies between aa's 622 and 634, while the NH(2) terminus of NBD1 lies between aa's 432 and 449. To facilitate biochemical studies of the expressed proteins, a Flag epitope was added to the NH(2) termini of full length CFTR, and of CFTR segments truncated before the normal COOH terminus (aa 1480). The functionally identified NBD1 boundaries are supported by Western blotting, coimmunoprecipitation, and deglycosylation studies, which showed that an NH(2)-terminal segment representing aa's 3-622 (Flag3-622) or 3-633 (Flag3-633) could physically associate with a COOH-terminal fragment representing aa's 634-1480 (634-1480); however, the latter fragment was glycosylated to the mature form only in the presence of Flag3-633. Similarly, 433-1480 could physically associate with Flag3-432 and was glycosylated to the mature form; however, 449-1480 protein seemed unstable and could hardly be detected even when expressed with Flag3-432. In excised-patch recordings, all functional severed CFTR channels displayed the hallmark characteristics of CFTR, including the requirement of phosphorylation and exposure to MgATP for gating, ability to be locked open by pyrophosphate or AMP-PNP, small single channel conductances, and high apparent affinity of channel opening by MgATP. Our definitions of the boundaries of the NBD1 domain in CFTR are supported by comparison with the solved NBD structures of HisP and RbsA.

Adenosine Monophosphate↗
PubMed2000

Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains.

Opening and closing of a CFTR Cl(-) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1-633 and 837-1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.

Adenosine Triphosphate↗