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Notes: [Auszug] We purified the nitrendipine receptor ~~ 500-fold to an apparent density of 2 nmol per mg protein. Gel electrophoresis of the purified receptor in the absence of a reducing agent yielded three major peptides of apparent Mr 142K, 56K and 3IK. Scans of the silver-stained gels indicate that 90% of the ...

Notes: Abstract Whole-cell Ca channel currents were recorded from guinea pig ventricular myocytes that were internally perfused with Cs solution and bathed in solutions containing 3.6 mM Ca, 3.6 mM Ba or 90 mM Ba (34° C). Single Ca channel currents were recorded from cell-attached membrane patches of similar myocytes; the patch pipettes contained a 90 mM Ba solution. 1. Although the shape of the whole-cellI–V relation was independent of the bathing solution, this was not the case with the location of the inward current maximum (V peak);V peak in 90 mM Ba was about 30 mV positive toV peak in 3.6 mM Ba. 2. The activation and inactivation of whole-cell currents were voltage dependent. Compared to the voltage dependencies in 3.6 mM Ba, those in 90 mM Ba were shifted by about 30 mV to the right, suggesting a neutralization of surface charges. 3. Observations compatible with the ion permeation model proposed by Hess and Tsien (1984) included (a) a depression of current during Ca/Ba solution exchange, (b) a high divalent to monovalent ion permeability, and (c) rectification of the outward limb of theI–V relation. 4. Estimated current densities atV peak were similar for myocytes in 3.6 mM Ca and 3.6 mM Ba, and about 10 times larger in 90 mM Ba. 5. Average currents (I *) calculated from ensembles of records of single Ca channel current had voltage-dependent time courses resembling those of whole-cellI Ba (90 mM). 6. Single-channelI *–V relations were superimposable on whole-cellI–V curves suggesting that voltage-dependent single-channel parameters (probability of opening, elementary current amplitude) can be related to the voltage-dependent macroscopic current parameters (activation, instantaneousI–V relation) when scaled by channel number. 7. The density of Ca channels in myocytes was calculated from whole-cellI Ba (90 mM) and average current through single channels. The outcome, 3–5 channels/μm2, agrees with two other recent estimates (Tsien et al. 1983; Lux and Brown 1984). However, it is difficult to reconcile with the much lower density that one would forecast from the frequency of functional channel observation in myocyte membrane patches (Pelzer et al. 1985c).

Notes: Abstract The possible role of electrostatic interactions for membrane binding and pore formation of annexin V has been analysed on the basis of a simple dielectric model. It is suggested that the binding of phospholipids to annexin V is regulated, at least initially, by the protein's electrostatic potential. The calculations show that a strong local gradient of the electrostatic potential exists at the membrane-protein interface and a membrane pore may be generated by electroporation. The observed specificity and regulation of ion conduction is suggested to reside in the protein part of the pore. On the basis of the three-dimensional structures of the protein and its hypothetical membrane complex, and electrophysiological measurements, a mechanical model of the transmembrane voltage regulation of the annexin's ion conduction properties is proposed.

Notes: Abstract. Whole-cell current recordings were used to examine the involvement of intracellular Ca2+ in the modulation of recombinant transient-receptor-potential like (TRPL) channels of Drosophila photoreceptor cells. TRPL was stably transfected in Chinese hamster ovary (CHO) cells and the expression of a calmodulin-binding protein with a molecular mass that corresponded to TRPL was demonstrated using calmodulin overlays. In cells expressing TRPL, ionic currents that were prominently outwardly rectifying were detected prior to activation of intracellular signalling pathways. The outwardly rectifying currents reversed close to 0 mV and did not occur after removal of permeant cations from the intracellular space. This suggests that TRPL forms non-selective cationic channels that appear to be constitutively active in mammalian cell lines. The TRPL channel currents were enhanced by manoeuvres that activate the phospholipase C (PLC) signalling pathway. These included activation of membrane receptors by thrombin, activation of G proteins by cell dialysis with guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) and release of Ca2+ from intracellular stores by dialysis with inositol 1,4,5-trisphosphate (IP3). After complete depletion of Ca2+ stores, IP3 had no effect on TRPL currents, suggesting that IP3 does not activate recombinant TRPL channels directly. However, thapsigargin, which induces a rise of cytosolic Ca2+, increased TRPL channel currents. Cell dialysis with solutions containing various concentrations of Ca2+ enhanced TRPL currents in a dose-dependent manner (EC50=450 nM Ca2+). Conversely, chelation of cytosolic Ca2+ abolished TRPL channel currents. The present results indicate that the activity of recombinant TRPL channels expressed in mammalian cell lines is up-regulated by a rise of cytosolic Ca2+.

Notes: Abstract. Single-channel current recordings were used to examine the properties and modulation of Drosophila transient-receptor-potential-like (TRPL) channels transiently expressed in HEK and COS cells. Recombinant TRPL channels were constitutively active and characterized by a conductance of 104 pS in on-cell membrane patches with 115 mM Na+ and 2 mM Mg2+ in the pipette solution. In inside-out membrane patches exposed to 115 mM Na+ plus 2 mM Mg2+, 115 mM Na+ plus 10 mM Mg2+, 90 mM Ca2+ and 90 mM Ba2+ on both sides, the single-channel conductances were 72 pS, 36 pS, 48 pS and 46 pS, respectively. The single TRPL channel currents reversed close to 0 mV and displayed a linear voltage dependence between –120 mV and +120 mV. Removal of cations from the pipette and bath solutions abolished inward and outward currents, respectively. Similar currents were not observed in mock-transfected and native cells. The opening probability of TRPL channels increased by depolarizing the membrane and accounted for the outward rectification of whole-cell TRPL currents. In on-cell membrane patches, the TRPL channel activity was enhanced by cell dialysis of 300 µM guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) and by a rise of intracellular Ca2+ (〉2 µM). Constitutively active TRPL channels depolarized the host cells to –10 mV and the membrane potential was restored by cell dialysis with 10 mM BAPTA. The present results suggest that TRPL forms non-selective cationic channels modulated by intracellular Ca2+ in mammalian cells.

Notes: Abstract (1) The Ca-channel gating behaviour during steady and stepwise depolarization was examined in recordings of single Ca-channel activity from cell-attached membrane patches of single ventricular cells isolated enzymatically from hearts of adult guinea pigs. The single-channel recordings were performed by means of the improved patch-clamp technique (Hamill et al. 1981) with 90 mM Ba in the pipettes. (2) Upon step depolarization, two types of current records were regularly observed in the ensembles: (1) traces with Ca-channel activity (in the form of closely-spaced brief pulses of inward current with a unitary amplitude) of various length, and (2) blank sweeps without any detectable single-channel opening. The records with Ca-channel activity show a distinct tendency for openings to occur towards the beginning of the clamp pulse, followed by long periods of silence. The blank sweeps seem to reflect a condition or conditions where the Ca channel is unavailable for opening. The corresponding ensemble mean currentI(t) displayed a rapid rising phase to its peak followed by a slow decay. (3) During steady depolarization, kinetic analysis of the distributions of all open and shut lifetimes revealed a monoexponential probability density distribution function of all open times. By contrast, more than two exponential terms were required for an accurate description of the frequency distribution of all shut lifetimes. Corresponding to the two well-separated fast closed time components, individual Ca-channel openings were grouped into bursts of openings. The bursting behaviour reflected fast gating transitions and was related to the fluctuations of the Ca channel between two short-lived closed states and one open state. This fast gating was terminated by the entrance of the Ca channel into at least one long-lived closed state, exit from which was slow in comparison to the rapid cycling. As consequence, bursts of openings were further grouped together in clusters of bursts, the cluster behaviour being related to slow gating transitions in the kinetics of the Ca channel. (4) The biphasic frequency distribution of the first latencies (resulting from the transit through the two short-lived shut states, before the open state is entered) superimposed on the first time derivative of the rising phase of the ensemble mean current,I(t), upon step depolarization. The time constant of the monoexponential distribution function of all cluster lifetimes matched the declining phase ofI(t) during maintained depolarization. Thus, the decrease of the probability of channel opening and the resulting decline ofI(t) seemed to be due to a transition of the Ca channel into the long-lived third class of shut state(s). (5) The responsiveness of the Ca channel in a series of trials decreased at positive holding potentials with a sigmoidal dependence on the potential of the conditioning depolarization due to an increasing number of blank single-channel current records within the ensembles. Traces with channel openings as well as blank sweeps tended to form sequences of consecutive single-channel current records upon conditioning depolarization. The Ca channel was only activatable, if a command pulse was applied during the occupancy of either the open state or the two short-lived shut states. If the long-lived shut state(s) was already occupied at the conditioning potential preceding the step depolarization, the Ca channel was unavailable for opening and a blank sweep was observed upon the voltage pulse. (6) A quantitative patch-to-patch variability in Ca-channel gating behaviour was detected. It was interpreted as the statistical deviation from the average kinetic behaviour of a single population of Ca channels. (7) The total time course of the ensemble mean currentI(t) was reconstructed by a convolution of the first latency and cluster lifetime distribution functions. The peak amplitude ofI(t) was mainly determined by the steady-state occupancy of the activatable states of the Ca channel. (8) Under comparable experimental conditions (90 mM external Ba), the pooled average behaviour of individual Ca channels in different membrane patches was the same as the bulk behaviour of all the Ca channels in the cardiac cell. (9) The fast and slow Ca-channel gating transitions are discussed in terms of a channel-state model where, according to lifetimes and transition rates, the respective channel states are divided into two subsets.

Notes: Abstract ICa was recorded in quinea pig ventricular myocytes using the whole-cell voltage-clamp technique. The shape of the I–V relation was unaffected by temperature (21–37°C) but there were large changes in ICa amplitude and time course. Steady-state responses indicated Q10 's of 2.96±0.14 (amplitude), 2.52±0.13 (time to peak), and 2.82±0.28 (T1/2 inactivation) (mean ± SD, n=6). Quick changes in temperature (T1/2〈30 s) induced pronounced deviations from the steady-state Q10 relations (early depression, compensatory overshoot). Thus, cardiac ICa differs from other currents in having a high amplitude-Q10 and an oscillatory response to rapid temperature changes.