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At least 595 records · Page 33Linked to original sources
PubMed1997

Estimating the number of channels in patch-clamp recordings: application to kinetic analysis of multichannel data from voltage-operated channels.

Important kinetic information of voltage-operated ion channels can be obtained by estimating the open probability, the availability, and the first latency, and by applying run analysis. In the case of multichannel patches, estimation of the number of available channels is a prerequisite for the above analysis. Here we describe a method for calculation of the a posteriori probability of the number of available channels in each sweep by using the Bayes formula. This probability serves as a measure for the number of channels and allows for first latency determination and run analysis. The methods described were applied to simulated and experimental data obtained from L-type Ca2+ channel recordings.

Bayes Theorem↗
PubMed1998

Patch clamp on the luminal membrane of exocrine gland acini from frog skin (Rana esculenta) reveals the presence of cystic fibrosis transmembrane conductance regulator-like Cl- channels activated by cyclic AMP.

Chloride channels in the luminal membrane of exocrine gland acini from frog skin (Rana esculenta) constituted a single homogeneous population. In cell-attached patches, channels activated upon exposure to isoproterenol, forskolin, or dibutyryl-cAMP and isobutyl-1-methyl-xanthine rectified in the outward direction with a conductance of 10.0 +/- 0.4 pS for outgoing currents. Channels in stimulated cells reversed at 0 mV applied potential, whereas channels in unstimulated cells reversed at depolarized potentials (28.1 +/- 6.7 mV), indicating that Cl- was above electrochemical equilibrium in unstimulated, but not in stimulated, cells. In excised inside-out patches with 25 mM Cl- on the inside, activity of small (8-pS) linear Cl--selective channels was dependent upon bath ATP (1.5 mM) and increased upon exposure to cAMP-dependent protein kinase. The channels displayed a single substate, located just below 2/3 of the full channel amplitude. Halide selectivity was identified as PBr > PI > PCl from the Goldman equation; however, the conductance sequence when either halide was permeating the channel was GCl > GBr >> GI. In inside-out patches, the channels were blocked reversibly by 5-nitro-2-(3-phenylpropylamino)benzoic acid, glibenclamide, and diphenylamine-2-carboxylic acid, whereas 4, 4-diisothiocyanatostilbene-2,2-disulfonic acid blocked channel activity completely and irreversibly. Single-channel kinetics revealed one open state (mean lifetime = 158 +/- 72 ms) and two closed states (lifetimes: 12 +/- 4 and 224 +/- 31 ms, respectively). Power density spectra had a double-Lorentzian form with corner frequencies 0.85 +/- 0.11 and 27.9 +/- 2.9 Hz, respectively. These channels are considered homologous to the cystic fibrosis transmembrane conductance regulator Cl- channel, which has been localized to the submucosal skin glands in Xenopus by immunohistochemistry (Engelhardt, J.F., S.S. Smith, E. Allen, J.R. Yankaskas, D.C. Dawson, and J.M. Wilson. 1994. Am. J. Physiol. 267: C491-C500) and, when stimulated by cAMP-dependent phosphorylation, are suggested to function in chloride secretion.

1-Methyl-3-isobutylxanthine↗
PubMed2004

Cilium-attached and excised patch-clamp recordings of odourant-activated Ca-dependent K channels from chemosensory cilia of olfactory receptor neurons.

It has previously been proposed that a Ca2+-dependent K+ conductance is implicated in the inhibitory odourant response in rat and toad olfactory receptor neurons. Previous whole-cell and single-channel measurements on inside-out excised patches, in addition to immunochemical evidence, indicated the presence of Ca2+-dependent K+ channels in olfactory cilia, the transducing structures of these sensory cells. Ca2+-dependent K+ channels opened in 'on-cilium' membrane patches from C. caudiverbera upon odourant stimulation. Furthermore, after excision in the inside-out configuration, the channel could be opened by micromolar Ca2+, in a Ca2+-dependent fashion, but it was unresponsive to cyclic AMP. We estimated that the Ca2+ concentration in the proximity of a Ca2+-dependent K+ channel within the cilia reaches at least 100 microM during the odour response. The K+ channel displayed a higher selectivity for K+ than for Na+. Our results support a role for this Ca2+-dependent K+ channel in chemotransduction.

Animals↗
PubMed2000

Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry.

Site-specific fluorescence recordings have shown great promise in understanding conformational changes in signaling proteins. The reported applications on ion channels have been limited to extracellular sites in whole oocyte preparations. We are now able to directly monitor gating movements of the intracellular domains of cyclic nucleotide-gated channels using simultaneous site-specific fluorescence recording and patchclamp current recording from inside-out patches. Fluorescence signals were reliably observed when fluorophore was covalently attached to a site between the cyclic nucleotide-binding domain and the pore. While iodide, an anionic quencher, has a higher quenching efficiency in the channel's closed state, thallium ion, a cationic quencher, has a higher quenching efficiency in the open state. The state and charge dependence of quenching suggests movements of charged or dipolar residues near the fluorophore during CNG channel activation.

Animals↗