1,721,028 research outputs found
Rates of diffusional exchange between small cells and a measuring patch pipette
No full textFluorescent compounds and specific ion currents (Na+, K+) were used to study the kinetics of the diffusional exchange between small cells and patch pipettes in the tight seal whole cell configuration of the patch clamp technique. (2) Changes in the intracellular concentration of the test substances following patch rupture could be fitted with single exponentials, provided the access resistanceR A of the pipette remained constant during diffusional equilibration. The diffusion time constants were linearly related to the access resistance. (3) When apparent diffusion rates were normalized with respect to access resistance they were found to be dependent on the cell size. However, the cell capacitance, which is proportional to the membrane area turned out not to be a precise measure of the cell size. (4) The experimental diffusion rates changed systematically with the aqueous diffusion coefficient and the inverse third root of the molecular weight. Linear interpolation with respect to these quantities provided estimates of diffusion time constants for the diffusion between patch pipettes and the cytoplasm for substances of interest
Identification of functionally important regions of the muscular chloride channel CIC-1 by analysis of recessive and dominant myotonic mutations
No full textMutations in the muscular voltage-dependent Cl- channel, CIC-1, lead to recessive and dominant myotonia, Here we analyse the effects of one dominant (G200R) and three recessive (Y150C, Y261C, and M485V) mutations after functional expression in Xenopus oocytes, Glycine 200 is a highly conserved amino acid located in a conserved stretch in the putatively cytoplasmic loop between domains D2 and D3, Similar to several other dominant mutations the amino acid exchange G200R leads to a drastic shift by similar to 65 mV of the open probability curve to more positive voltages, As explored by co-expression studies, the shift is intermediate in heteromeric mutant/WT channels, Open channel properties such as single channel conductance, rectification or ion selectivity are not changed, Thus we identified a new region of the CIC-1 protein in which mutations can lead to drastic shifts of the voltage dependence, The recessive mutation M485V, which is located in a conserved region at the beginning of domain D10, leads to a drastic reduction of the single channel conductance from 1.5 pS for WT to similar to 0.3 pS, In addition, the mutant is strongly inwardly rectifying and deactivates incompletely at negative voltages, ion-selectivity, however, is unchanged, These electrophysiological properties fully explain the recessive phenotype of the mutation and identify a new region of the protein that is involved in ion permeation and gating of the CIC-1 channel, The other two recessive mutations (Y150C and Y261C) had been found in a compound heterozygous patient, Surprisingly, expression of these mutants in oocytes yielded currents indistinguishable from WT CIC-1 when explored by two-electrode voltage clamp recording and patch clamping (either singly or both mutations co-expressed), Other mechanisms that are not faithfully represented by the Xenopus expression system must therefore be responsible for the myotonic symptoms associated with these mutations
Properties of voltage-gated chloride channels of the ClC gene family
No full textWe review the properties of ClC chloride channels, members of an expanding gene family originally discovered by the cloning of the ClC‐0 chloride channel from Torpedo electric organ. There are at least nine different ClC genes in mammals, several of which seem to be expressed ubiquitously, while others are expressed in a highly specific manner (e.g. the muscle‐specific ClC‐1 channel and the kidney‐specific ClC‐K channels). The newly cloned rat ClC‐4 is strongly expressed in liver and brain, but also in heart, muscle, kidney and spleen. ClC chloride channels are structurally unrelated to other channel proteins and have twelve putative transmembrane domains. They function as multimers with probably four subunits. Functional characterization is most advanced with ClC‐0, ClC‐1 (mutations which cause myotonia) and ClC‐2, a swelling‐activated chloride channel. Many of the new ClC family members cannot yet be expressed functionally
Activation and Inactivation of Homomeric KvLQT1 Potassium Channels
No full textAbstractThe voltage-gated potassium channel protein KvLQT1 (Wang et al., 1996. Nature Genet. 12:17–23) is believed to underlie the delayed rectifier potassium current of cardiac muscle together with the small membrane protein minK (also named IsK) as an essential auxiliary subunit (Barhanin et al., 1996. Nature. 384:78–80; Sanguinetti et al., 1996. Nature. 384:80–83). Using the Xenopus oocyte expression system, we analyzed in detail the gating characteristics of homomeric KvLQT1 channels and of heteromeric KvLQT1/minK channels using two-electrode voltage-clamp recordings. Activation of homomeric KvLQT1 at positive voltages is accompanied by an inactivation process that is revealed by a transient increase in conductance after membrane repolarization to negative values. We studied the recovery from inactivation and the deactivation of the channels during tail repolarizations at −120mV after conditioning pulses of variable amplitude and duration. Most measurements were made in high extracellular potassium to increase the size of inward tail currents. However, experiments in normal low-potassium solutions showed that, in contrast to classical C-type inactivation, the inactivation of KvLQT1 is independent of extracellular potassium. At +40mV inactivation develops with a delay of 100ms. At the same potential, the activation estimated from the amplitude of the late exponential decay of the tail currents follows a less sigmoidal time course, with a late time constant of 300ms. Inactivation of KvLQT1 is not complete, even at the most positive voltages. The delayed, voltage-dependent onset and the incompleteness of inactivation suggest a sequential gating scheme containing at least two open states and ending with an inactivating step that is voltage independent. In coexpression experiments of KvLQT1 with minK, inactivation seems to be largely absent, although biphasic tails are also observed that could be related to similar phenomena
Washout phenomena in dialyzed mast cells allow discrimination of different steps in stimulus-secretion coupling
No full textTransient increases of intracellular calcium and exocytotic activity of rat peritoneal mast cells following stimulation with compound 48/80 were monitored using the Ca-indicator dye fura-2 and the capacitance measurement technique. It is known that mast cells very rapidly lose their secretory response towards antigenic or compound 48/80-induced stimulation in the whole-cell recording configuration of the patch-clamp technique due to “washout” of signal mediators. In contrast, we found that calcium transients remained unaffected by intracellular dialysis for as long as 10 min. The fast “washout” phenomenon of exocytosis could be overcome by supplementing the pipette filling solution with guanosinetriphosphate (GTP) indicating a major role for GTP-binding proteins in secretion. The restoration of exocytosis was transient and decayed within three minutes, suggesting diffusional escape of one or several other cytoplasmic substances involved in stimulus-secretion coupling. Quantitative aspects of this process and the implications of its differential effects on Ca-transients versus secretion are discussed.Transient increases of intracellular calcium and exocytotic activity of rat peritoneal mast cells following stimulation with compound 48/80 were monitored using the Ca-indicator dye fura-2 and the capacitance measurement technique. It is known that mast cells very rapidly lose their secretory response towards antigenic or compound 48/80-induced stimulation in the whole-cell recording configuration of the patch-clamp technique due to “washout” of signal mediators. In contrast, we found that calcium transients remained unaffected by intracellular dialysis for as long as 10 min. The fast “washout” phenomenon of exocytosis could be overcome by supplementing the pipette filling solution with guanosinetriphosphate (GTP) indicating a major role for GTP-binding proteins in secretion. The restoration of exocytosis was transient and decayed within three minutes, suggesting diffusional escape of one or several other cytoplasmic substances involved in stimulus-secretion coupling. Quantitative aspects of this process and the implications of its differential effects on Ca-transients versus secretion are discussed
The human two-pore channel 1 is modulated by cytosolic and luminal calcium
No full textAbstractTwo-pore channels (TPC) are intracellular endo-lysosomal proteins with only recently emerging roles in organellar signalling and involvement in severe human diseases. Here, we investigated the functional properties of human TPC1 expressed in TPC-free vacuoles from Arabidopsis thaliana cells. Large (20 pA/pF) TPC1 currents were elicited by cytosolic addition of the phosphoinositide phosphatidylinositol-(3,5)-bisphosphate (PI(3,5)P2) with an apparent binding constant of ~15 nM. The channel is voltage-dependent, activating at positive potentials with single exponential kinetics and currents are Na+ selective, with measurable but low permeability to Ca2+. Cytosolic Ca2+ modulated hTPC1 in dual way: low μM cytosolic Ca2+ increased activity by shifting the open probability towards negative voltages and by accelerating the time course of activation. This mechanism was well-described by an allosteric model. Higher levels of cytosolic Ca2+ induced a voltage-dependent decrease of the currents compatible with Ca2+ binding in the permeation pore. Conversely, an increase in luminal Ca2+ decreased hTPC1 activity. Our data point to a process in which Ca2+ permeation in hTPC1 has a positive feedback on channel activity while Na+ acts as a negative regulator. We speculate that the peculiar Ca2+ and Na+ dependence are key for the physiological roles of the channel in organellar homeostasis and signalling.</jats:p
Small Molecules Targeting Kidney ClC-K Chloride Channels: Applications in Rare Tubulopathies and Common Cardiovascular Diseases
Full text linkGiven the key role played by ClC-K chloride channels in kidney and inner ear physiology and pathology, they can be considered important targets for drug discovery. Indeed, ClC-Ka and ClC-Kb inhibition would interfere with the urine countercurrent concentration mechanism in Henle’s loop, which is responsible for the reabsorption of water and electrolytes from the collecting duct, producing a diuretic and antihypertensive effect. On the other hand, ClC-K/barttin channel dysfunctions in Bartter Syndrome with or without deafness will require the pharmacological recovery of channel expression and/or activity. In these cases, a channel activator or chaperone would be appealing. Starting from a brief description of the physio-pathological role of ClC-K channels in renal function, this review aims to provide an overview of the recent progress in the discovery of ClC-K channel modulators
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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