Abstract: Using the patch-clamp method, we studied the influence of external alkali and organic monovalent cations on the single-channel properties of the adenosine triphosphate (ATP)-activated recombinant human P2X(7) receptor. The slope conductance of the hP2X(7) channel decreased and the reversal potential was shifted to more negative values as the ionic diameter of the organic test cations increased. From the relationship between single-channel conductance and the dimensions of the inward current carrier, the narrowest portion of the pore was estimated to have a mean diameter of approximately 8.5 A. Single-channel kinetics and permeation properties remained unchanged during receptor activation by up to 1 mM ATP(4-) for >1 min, arguing against a molecular correlate of pore dilation at the single P2X(7) channel level. Substitution of extracellular Na(+) by any other alkali or organic cation drastically increased the open probability of the channels by prolonging the mean open time. This effect seems to be mediated allosterically through an extracellular voltage-dependent Na(+) binding site with a K(d) of approximately 5 mM Na(+) at a membrane potential of -120 mV. The modulation of the ATP-induced hP2X(7) receptor gating by extracellular Na(+) could be well described by altering the rate constant from the open to the neighboring closed state in a C-C-C-O kinetic receptor model. We suggest that P2X(7) receptor-induced depolarization and associated K(+)-efflux may reduce Na(+) occupancy of the regulatory Na(+) binding site and thus increase the efficacy of ATP(4-) in a feed-forward manner in P2X(7) receptor-expressing cells.
Abstract: Human P2X7 receptors were expressed in Xenopus laevis oocytes and single channels were recorded using the patch-clamp technique in the outside-out configuration. ATP4- evoked two types of P2X7 receptor-mediated single channel currents characterized by short-lived and long-lived openings. The short- and long-lasting open states had mean open times of approximately 5 and approximately 20 ms and slope conductances near -60 mV of 9 and 13 pS, respectively. The open probabilities of the short and long openings were strongly [ATP4-]-dependent with EC50 values of approximately 0.3 mM and approximately 0.1 mM ATP4-, respectively. The channel kinetics did not change significantly during sustained P2X7 receptor activation for several minutes, as was also observed in recordings in the cell-attached patch-clamp configuration. Activation and deactivation of the short openings followed exponential time courses with time constants in the range of 20 ms, and displayed a shallow [ATP4-] dependence of the activation process. The kinetics of the short channel openings at negative membrane potentials fitted well to a linear C-C-C-O model with two ATP4- binding steps at equal binding sites with a dissociation constant Kd of 139 microM.
Abstract: To characterize ion channels expressed in cell membrane of human keratinocytes, patch-clamp recordings were carried out in HaCaT cells. Two types of large-conductance K(+) channels (about 250 pS) were measured. One type was activated by micromolar concentrations of intracellular Ca(2+) ions ([Ca(2+)](i)) and membrane depolarization, the other was [Ca(2+)](i) independent. The channels were neither dependent on intracellular ATP nor Mg(2+) nor on membrane stretch. We conclude that HaCaT keratinocytes express Ca(2+)-dependent maxi K(+) channels and still unknown large Ca(2+)-independent K(+) channels. These K(+) channels may affect the proliferation and differentiation of human keratinocytes by the influence on the resting potential, which may control the Ca(2+) influx across the cell membrane.
Abstract: Purinergic P2X(1) and P2X(7) receptors are co-expressed in several cell types such as lymphocytes or epithelial cells. Here we examined whether these two P2X subtypes interact with each other in a manner that results in a mutual alteration of their electrophysiologic behaviour. Furthermore, since specific pharmacological tools are needed to assign distinct effects to a particular receptor subtype in native cells, we assessed a series of compounds for their capacity to separate individual current components in cells that co-expressed both receptor subtypes. In Xenopus oocytes, co-expression neither changed the time courses of activation, desensitization and deactivation nor recovery from desensitization when compared to oocytes that express either hP2X(1) or hP2X(7) receptors alone. A selective activation of hP2X(7) receptors was achieved with benzoyl-benzoyl-ATP, which did not activate P2X(1) receptor currents. P2X(7) receptors could also be selectively activated by ATP when co-applied with 1 microM NF449, a suramin derivative, which is 100,000 fold more potent in blocking P2X(1) than P2X(7) receptors. alphabeta-methylene-ATP, a reportedly hP2X(1) receptor-specific agonist, as well as oxidized-ATP, brilliant blue or KN62, reported hP2X(7) receptor antagonists, were found to be ineffective in separating hP2X(1) receptor current from the P2X(7) current. The best way for a selective activation of the hP2X(1) receptor component in cells co-expressing the P2X(7) receptor is the application of low concentrations of ATP (< 1 microM) or the addition of Mg2+ when using higher concentrations of ATP.
Abstract: The antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were analyzed at homomeric human P2X(1) and P2X(7) receptor subtypes (hP2X(1) and hP2X(7)) heterologously expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. At activating ATP concentrations of 1 microM (hP2X(1)) and 100 microM (hP2X(7)), IC(50) values of 0.05 nM and 40 microM were found for hP2X(1) and hP2X(7) receptors, respectively. The Schild analysis revealed a pA(2) of 10.7 at hP2X(1). Wash-in and wash-out of 10 nM NF449 were nearly complete within 16 s and 4 min, respectively, at the hP2X(1) receptor. An increase in the activating ATP concentration to 100 microM shifted the NF449 concentration-inhibition curve rightwards for the hP2X(1) receptor. NF449 decelerated activation as well as desensitization of hP2X(1). It is concluded that NF449 acts as a reversible competitive antagonist at the hP2X(1) with much higher potency at hP2X(1) than at hP2X(7) receptors. NF449 may hence be excellently suited to discriminate between both receptors in native human tissues.
Abstract: A glutamate to alanine exchange at amino acid position 496 of the human P2X(7) receptor was recently shown to be associated with a loss of function in human B lymphocytes in terms of ATP-induced ethidium(+) uptake, Ba(2+) influx, and induction of apoptosis (Gu BJ, Zhang WY, Worthington RA, Sluyter R, Dao-Ung P, Petrou S, Barden JA, and Wiley JS. J Biol Chem 276: 11135-11142, 2001). Here we analyzed the effect of the Glu(496) to Ala exchange on the channel properties of the human P2X(7) receptor expressed in Xenopus oocytes with the two-microelectrode voltage-clamp technique. The amplitudes of ATP-induced whole cell currents characteristic of functional expression, kinetic properties including ATP concentration dependence, and permeation behavior were not altered by this amino acid exchange. Also in HEK293 cells, the Ala(496) mutant mediated typical P2X(7) receptor-dependent currents like the parent Glu(496) hP2X(7) receptor. Because the function of the P2X(7) receptor as an ATP-gated channel for small cations including Ba(2+) remained unaffected by this mutation, we conclude that Glu(496) plays a critical role in pore formation but does not determine the ion channel properties of the human P2X(7) receptor.
Abstract: Patch-clamp recordings were carried out in the inside-out configuration in human keratinocytes of the cell line HaCaT. Patch pipettes were filled with 150 mM KCl, 1 mM CaCl(2) and 10 mM HEPES. In symmetrical KCl solutions, single channel currents from a large conductance channel (about 170 pS) were measured. Replacement of 120 mM KCl by K-aspartate had only a minor influence on the single channel conductance and on the reversal potential. In intracellular solution in which K(+) has been replaced by Na(+) or NMDG(+), the reversal potential shifted to > + 40 mV indicating K(+) as the main charge carrier. The channels were neither dependent on intracellular Ca(2+) (between 0.8 nM and 10 micro M), ATP (at 0 and 1 mM) nor Mg(2+) (at 0 and 0.5 mM). The mean current showed an outward rectification that can be mainly attributed to the voltage dependence of the open probability. The channels displayed bursting kinetics with a mean open time of about 2 ms and closed times of about 0.2, 2 and 20 ms. The mean open probability was usually low (0.05) but increased occasionally (0.6) mainly due to a lower probability of long closings. We conclude that these K(+) channels contribute to the resting potential of human keratinocytes which may control the Ca(2+) influx and thereby their proliferation and differentiation.
Abstract: The kinetics of the intracellular Ca2+ concentration ([Ca2+]i) of vascular smooth muscle cells (VSMCs) in rat small mesenteric arteries was investigated by confocal laser scanning microscopy using the fluorescent Ca2+ indicator fluo-3 AM. One micromole noradrenaline (NA) induced randomly distributed transient elevations of [Ca2+]i in several single VSMCs which were weakly temporally coupled. Higher NA concentrations of 3 or 10 microM, however, induced strongly synchronised [Ca2+]i oscillations in VSMCs. In preparations with intact endothelium, the synchronisation of [Ca2+]i signals was attenuated by acetylcholine (ACh) but augmented by the NO synthase antagonist L-NAME, pointing to a desynchronising effect of the endothelium even under basal conditions. In preparations with or without intact endothelium sodium nitroprusside (SNP) as well as the gap-junction uncoupler heptanol reversibly desynchronised the [Ca2+]i transients. The effect of ACh but not that of SNP was influenced by L-NAME. Propagated intracellular [Ca2+]i waves had a velocity of 25 microm/s. The phase shift of [Ca2+]i oscillations between single VSMCs were maximally 2s and independent of the distance of up to 90 microm between individual cells. Therefore, we consider intercellular [Ca2+]i waves to be too slow to account for the synchronisation of [Ca2+]i oscillations.We conclude that the coupling of [Ca2+]i signals in vascular smooth muscle cells is not constant but highly regulated by NA and by endothelium derived NO. Oscillations of vessel contraction at high sympathetic tone may be induced by synchronisation of [Ca2+]i transients of distinct VSMCs whereas endothelium derived NO inhibits vasomotion by desynchronising [Ca2+]i transients of single VSMCs.
Abstract: Despite a high Ca2+ -permeability of the P2Z receptor in human B lymphocytes, extracellular ATP4- has only a minor effect on global [Ca2+]i. The aim of this study was to reveal the mechanisms responsible for this discrepancy. We investigated the relationship between ATP4- -application, Cai 2+ -response, membrane current and membrane potential in two human B cell lines and in human tonsillar B cells. This was achieved by a combination of FACS- and voltage clamp measurements and the usage of appropriate voltage- and Ca2 -sensitive fluorescent dyes. ATP4 -induced changes in whole-cell current and [Ca2]i were blocked by extracellular as well as intracellular Na+. Under current clamp conditions, ATP4- -induced Na+ -entry diminished the Ca2+ entry via reduction of the driving force. A substantial increase in [Ca2+]iinduced by ATP4- was only observed in Na+ -free solutions.The pathway of signal transduction activated by ATP4via P2Z receptor of human B lymphocytes under physiological conditions seems not to operate by an increase in the global intracellular Ca2+ -concentration, but rather by the depolarization of the cell membrane as a result of the Na+-influx.
Abstract: 1. The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X(7) receptor (hP2X(7)), an N-terminally hexahistidyl-tagged hP2X(7) receptor (His-hP2X(7)), or a truncated His-hP2X(7) receptor (His-hP2X(7)DeltaC) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique. 2. The activation time course of the wild-type hP2X(7) receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component. 3. The amplitude of the exponentially activating current component of the wild-type hP2X(7) receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity non-cooperative activation sites with apparent dissociation constants of about 4 and 200 microM free ATP(4-), respectively. 4. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 microM. 5. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X(7) or His-hP2X(7)DeltaC. 6. Our data indicate that the hP2X(7) receptor possesses at least two types of activation sites, which differ in ATP(4-) sensitivity by a factor of 50. The degree of occupation of these two sites influences both activation and deactivation kinetics. Both N- and C-terminal domains appear to be important determinants of the current elicited by activation of the sites with low ATP sensitivity, but not for that mediated by the highly ATP-sensitive sites.
Abstract: Human B lymphocytes express an ATP-gated ion channel (P2Z receptor), which shares similarities with the recently identified P2X7 receptor. Using gene specific primers, we have now isolated P2X7 cDNA from the total RNA of human B lymphocytes. This hP2X7 receptor subtype was expressed in Xenopus oocytes and electrophysiologically characterized. The hP2X7 receptor is similar to, but does not completely match, P2Z of human B cells. The hP2X7 receptors resemble the P2Z receptors with regard to the ATP concentration of half maximal activation, reproducibility, permeation characteristics and lack of desensitization of the ATP-evoked currents. However, in contrast to the native lymphocytic P2Z receptor, the time course of activation of hP2X7 displayed an additional linearly increasing current component. Furthermore, a second, small and slowly deactivating current component exists only in hP2X7 expressed in oocytes. The activation and deactivation kinetics as well as permeation characteristics of hP2X7 are different from rat P2X7 recently expressed in oocytes. Unlike in mammalian cells, hP2X7 expressed in Xenopus oocytes is not sufficient to induce large non-selective pores.
Abstract: The effect of the suramin analogue 8,8'-(carbonylbis(imino-4, 1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3 , 5-naphthalenetrisulfonic acid) (NF279) was analyzed on human P2X(1) and P2X(7) receptor subtypes (human P2X(1) and human P2X(7)) heterologously expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. At activating ATP concentrations of 1 microM (human P2X(1)) and 10 microM ATP (human P2X(7)), IC(50) values of 0.05 microM and 2.8 microM were found for human P2X(1) and human P2X(7) receptors, respectively. An increase in the activating [ATP] shifted the NF279 concentration-inhibition curve rightwards for both receptors. NF279 slowed the activation of both human P2X(1) and human P2X(7) as well as the desensitization of human P2X(1). The data support a model in which desensitization of P2X(1) is dependent on preceding activation of these P2X receptors. It is concluded that NF279 acts as a competitive antagonist with much higher potency at human P2X(1) than at P2X(7) receptors. NF279 may hence be suited to discriminate between both receptors in native tissues.
Abstract: Single channel properties of human P2X4 receptors expressed in human embryonic kidney cells have been investigated by outside-out mode patch clamp recordings. P2X4 channel activity was characterized by very fast kinetics. The current-voltage relationship was strongly non-linear at potentials <-100 mV. A slope conductance of approximately 9 pS was estimated at the approximately linear part of the current-voltage relation (>-100 mV). External Mg2+ reversibly decreased the amplitude of ATP-evoked single channel currents in a concentration-dependent manner but independent of the membrane potential. Additionally, extracellular Mg2+ shortened the mean open time whereas the mean closed time was not affected. Thus, Mg2+ ions are proposed to inhibit the function of human P2X4 receptors by means of an open-channel block with a Mg2+ binding site at the exterior surface of the pore.
Abstract: The living, not-cornified part of the epidermis consists mainly of keratinocytes. The control mechanisms of proliferation and differentiation are only partly understood. Similarly, the influence of ionic channels of the cell membrane on the proliferation of keratinocytes remains unclear. Preliminary investigations point to a relation between the opening of ionic channels and keratinocyte proliferation. Therefore, voltage clamp experiments were performed to gain further knowledge of the electrophysiological characteristics of human keratinocyte cellular membranes. In-vitro cultured keratinocytes of the cell line HaCaT were characterized by means of the voltage-clamp technique. As measured in the whole-cell configuration, changing the extracellular K+ or Cl--concentration shifted the membrane potential of HaCaT cells. Application of the patch-clamp-technique in the cell-attached and inside-out configuration revealed an ionic channel with a conductance of multiples of 200 pS. The reversal potential of the single channel current was shifted by substituting of intracellular Cl- by aspartate-. Channel openings disappeared after addition of 0.1 mM of the anion channel blocker 4',4' diisothiocyanato-stilbene-2-2' disulfonic acid (DIDS). It is concluded that this channel contributes to the Cl--conductance of the cellular membrane and is a determinant of the membrane potential of human keratinocytes. This channel may represent a target for pharmacological manipulation of the membrane potential and possibly the growth of human keratinocytes in dermatological proliferation disorders.
Abstract: The rapid application system described has been used to study a variety of ionic channels in several different types of single cells. The system is inexpensive, easy to install, and can be used repeatedly. The consumption of UTS, i.e., drugs or agonists, is low. The time interval between switching the valve and the expected effect is often shorter than 150 ms for cells about 8-15 microns in diameter and is about 20-25 ms for patches positioned in the hole of the U-tube. Time and duration of substance application can be controlled by a computer connected to a digital-analog (D/A) output.
Abstract: The proliferation of in vitro cultivated keratinocytes from the HaCaT cell line while under the influence of different ion channel blockers, i.e. calcium channel blockers tetraethylammonium (TEA) and the nonspecific anion channel blocker 4',4'-diisothiocyanato-stilbene-2-2' disulfonic acid (DIDS) was investigated. There were changes in proliferation of the keratinocytes brought on by TEA and DIDS. It can be assumed that the effects of TEA on proliferated keratinocytes lead to a temporary arrest of cells in the G(1)-phase of the cell cycle. The change in the extracellular K+ concentration ([K+]e) had no influence on the proliferation of keratinocytes. During the entire course of investigations, the adding of DIDS leads to an increase in proliferation. Proliferation of keratinocytes is known to be greatly dependent on intracellular Ca2+ content ([Ca2+]i). The blocking of ion channels with TEA or DIDS does not lead to a change in the free [Ca2+]i. Also a change in the [K+]e does not lead to a displacement of the [Ca2+]i of keratinocytes.
Abstract: The effects of N-(dicyclohexyl-carbamoylmethyl)-N-(3-diethylamino-propyl)-4-nitro -benzamide hydrochloride (AWD 23-111), a novel antiarrhythmic compound, were studied in isolated cardiomyocytes of guinea pigs. Using whole-cell configuration of the patch-clamp technique AWD 23-111 was tested for its ability to block the delayed rectifier potassium channel (IK). In guinea pig ventricular myocytes the current is composed of two components: IKr, a rapidly activating current and IKs, a slowly activating component which were discriminated by their different activation and deactivation behaviour. In this preparation AWD 23-111 displayed concentration dependent inhibitory effects on IKr as well as on IKs in the tested concentration range between 1 and 100 mumol/l. This blocking effect was independent of the stimulation frequency (0.2, 1 and 2 Hz). There was no influence of AWD 23-111 on the amplitude of L-type calcium whole-cell currents. The compound significantly prolonged action potential duration (APD) at a stimulation frequency of 2 Hz (1 and 10 mumol/l). At 0.2 Hz there was no effect on APD. Our results suggest that AWD 23-111 blocks both components of IK without a reverse use-dependent effect on APD which limits the therapeutic potential of most other class III agents.
Abstract: 1. The mechanism of the sustained acetylcholine-induced endothelium-dependent hyperpolarization (EDH) in intact rat small mesenteric arteries prestimulated with noradrenaline (10(-6) M) was investigated by means of the single microelectrode voltage-clamp method. 2. The vascular smooth muscle cells (VSMCs) in this preparation are poorly or even not coupled for the reasons that: (1) the mean input resistance Rlnp of the clamped vascular smooth muscle increases from 120 M omega under control conditions to 440 M omega after application of K+ channel blocking drugs, (2) the voltage relaxation after injection of hyperpolarizing currents has a monoexponential time course and is linearly dependent on Rlnp, and (3) voltage steps induced by current-clamp steps are not transferred to locations in the vascular musculature 120 microns apart from the current injecting microelectrode. 3. Sustained (> 5 min) application of ACh (10(-5) M) hyperpolarized the VSMCs by induction of a hyperpolarizing current. This effect was completely blocked by the inhibitor of the nitric oxide (NO) synthase L-NAME (10(-3) M) but not by the inhibitor of the soluble guanylate cyclase (sGCl) Methylene Blue (MB, 10(-4) M). 4. Application of the NO donor sodium nitroprusside (SNP, 10(-6) M) for more than 5 min mimicked the induction of the endothelium-dependent hyperpolarizing current in vessels with destroyed endothelium. The reversal potential of this current is dependent on the extracellular K+ concentration. The effect of SNP could also not be blocked by MB. 5. The blockers of ATP-dependent and Ca(2+)-dependent K+ channels, glibenclamide (Glb, 10(-5) M) and charybdotoxin (CTX, 5 x 10(-8) M), respectively, blocked a hyperpolarizing current in the VSMCs similar to the ACh- or SNP-induced current. 6. The isolated application of either Glb or CTX did not block the activation of the hyperpolarizing current by SNP. Only the combined administration of Glb and CTX blocked the SNP-induced current completely. 7. Our results suggest that in rat small mesenteric artery, ACh hyperpolarizes the VSMCs tonically by activating both ATP- and Ca(2+)-dependent K+ currents, only via release of NO from the endothelium without need for activation of the sGCl.
Abstract: 1. Using the patch clamp method in the outside-out configuration, purinoceptor-dependent unitary currents were measured in tonsillar and transformed tonsillar human B lymphocytes. 2. Single channel currents were evoked by ATP4-, the free-acid form of ATP, and by 2',3' O-benzoyl-4-benzoyl-ATP (BzATP) in the micromolar concentration range, but not by 10 mM ADP3- or 0.5 mM Mg(2+)-bound ATP. 3. The channels could be activated and deactivated several times for as long as 30 min even in the absence of intracellular ATP, GTP, or glucose. 4. The channels were selective for small cations and had a conductance of 9 pS with Cs+ as the intracellular and Na+ as the extracellular monovalent cation. 5. The half-maximal activation of the channels was obtained by 114 microM ATP4- and by 16 microM BzATP. The increase in the open probability after raising the ATP4- concentration was mainly due to a decrease in the times the channels spend in the closed state. 6. It is concluded that human B lymphocytes possess cationic channels directly gated by extracellular ATP4-. Their agonist binding characteristics are typical for P2z purinoceptors, but their permeation behaviour is different from the large non-specific pores formed by ATP4- in fibroblasts, macrophages and mast cells.
Abstract: Concentrations of 1-4 mumol l-1 isoproterenol cause both in right ventricular papillary muscles and in enzymatically isolated myocytes of the guinea-pig a Ca2+ overload-induced state which is functionally characterized by biphasic (multiphasic) twitches and biphasic (multiphasic) intracellular calcium transients, respectively, during excitation-contraction coupling. This state was stabilized in the in vitro experiments for some hours by a co-ordination of the interstimulus interval, the temperature of the superfusion fluid and the addition of calcium agonists. The functional stability is the precondition for the reproducibility of the experimental results particularly after the application of long-lasting stimulation programmes. Changes in the shape of biphasic contractions were compared with changes in the time course of biphasic intracellular calcium transients using three manipulations of a different kind: (1) the interruption of the steady pacing rhythm, (2) the variation of the interstimulus interval, (3) the addition of ryanodine. It was shown that: (1) The BOWDITCH staircase in calcium overloaded multicellular preparations is changed in that each individual component of the twitch passes through its own staircase. A homologous behaviour can be observed in the configuration of the phasic and tonic component of biphasic intracellular calcium transients. (2) At different driving frequencies the relative proportion of the two components of a biphasic twitch corresponds to the time integrals of the two components of biphasic intracellular calcium transients. (3) Ryanodine suppresses both the first component of the biphasic twitch and the phasic component of the biphasic intracellular calcium transient. The SR Ca(2+)-ATPase inhibitor thapsigargin increases the second component of the biphasic calcium transient. This supports the hypothesis that the size of the tonic component is in part determined by intracellular calcium reuptake. The results of both kinds of experiments would be compatible with the assumption that in calcium overloaded mammalian cardiac cells calcium reaches the contractile system directly as well as via two intracellular stores ('extended two-Ca-store concept').
Abstract: Adenosine 5'-triphosphate-(ATP)-induced whole-cell currents were studied in human B-lymphocytes, transformed by the Epstein-Barr virus, by means of the tight-seal voltage-clamp technique. During bath application of ATP, the membrane conductance was increased. The change of membrane conductance occurred within milliseconds. The dose response relationship for the ATP(4-)-elicited membrane current (Ip) was fitted by the Hill function with a Hill coefficient of 1 and a KD value of 0.2 mmol/l. Adenosine, as well as the Mg(2+)-bound form of ATP, did not effect the membrane conductance. Ip did not desensitize within 1 min and could be evoked repeatedly up to 100 times in 1 cell in the presence of the G-protein blocker Guanosine 5'-o-(2-thiodiphosphate) (GDP [beta S]). Therefore, it seems that ion channels in form of P2Z-purinoceptors are involved in the observed effects. The permeability (P) sequence for cations carrying Ip was PCa:PK:PCs:PNa:PTRIS = 35:2:1.2:1:0.1. The reversal potential of IP was not changed by substitution of intracellular Cl- for aspartate, indicating that anions are not involved in the purinoceptor-dependent conductance. A single-channel conductance of P2Z-receptor-dependent ion channels of about 3 pS was determined by noise analysis of Ip.
Abstract: Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started a approximately 10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37 degrees C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.
Abstract: Single calcium channel (Ca channel) currents were measured using the patch-clamp technique in isolated ventricular myocytes of the frog (Rana esculenta). Sodium was used as the charge carrier. After formation of cell-attached patches, the proteolytic enzyme thrombin was added to the bath solution, where it increased the amplitude of the averaged currents more than twofold, by decreasing the number of empty sweeps and reducing the time constant of the slow exponential term of the shut-time histogram. Single channel conductance was not changed by thrombin. If the activation kinetics of the Ca channels are described by the commonly used C1-C2-O model, where C1 and C2 indicate closed states 1 and 2 respectively and O denotes the open state, thrombin increases the open-state probability in the non-empty sweeps by increasing the rate constant (k1) for the transition from C1 to C2. It is shown that thrombin acts via an H-7 blockable pathway.
Abstract: The pharmacological modification of the thrombin effect on the mechanical and electrical responses of frog heart was examined in the Straub heart preparation and in single ventricular cells. Associated with the positive inotropic action thrombin increases voltage and duration of action potentials of isolated frog ventricular cells. As found by the patch-clamp technique in the cell-attached mode, thrombin stimulates single L-type Ca2+ channels, presumably mediated by a second messenger. The enhancement of contractility by thrombin depends on the proteolytic activity of the enzyme because enzymatically inactivated thrombin has no effect on frog hearts. The positive inotropic effect of thrombin as well as its stimulation of Ca2+ channel currents were inhibited by the protein kinase C blocker 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H7). However, phorbol 12-myristate 13-acetate (PMA), a known stimulator of protein kinase C, was ineffective in stimulating the inotropic action of thrombin. The inhibition of the thrombin-induced enhancement of contractility by indometacin indicates an involvement of arachidonic acid in the action of thrombin on frog heart.
Abstract: Run down of calcium channel currents carried by Ba2+ was studied in enzymatically isolated ventricular cells from guinea-pig hearts using the suction pipette method. The decay of the Ba currents elicited by 100 ms voltage steps from -60 to 0 mV could be best described biexponentially. The peak current amplitude declined monotonously during dialysis of cell interior, whereas the inactivation showed a biphasic behaviour. After start of intracellular perfusion, the time constants of the fast and slow exponential component decreased up to the 6th and 10th minute, respectively. Thereafter the inactivation decelerated. These changes of the inactivation behaviour could be described by a model of current dependent inactivation. It was assumed, that in the first phase the decay is accelerated due to predominating of washout of phosphorylating agents and in the second phase the reduced conductance of the cell membrane for Ba2+ due to progressive irreversible closure of Ca channels causes slowing down of inactivation.
Abstract: Effects of the proteolytic enzyme thrombin in the modulation of cardiac Ca-channel currents were examined in single ventricular cells from frog myocardium, using the whole-cell voltage clamp technique (1). Application of 3.8 . 10(-9) M thrombin to the bath increased the peak of the Ca-channel current by 84 +/- 35% (8 cells). Hirudin (31.10(-9)M), a specific thrombin inhibitor, blocked the thrombin-induced increase of this current. The increase in the current can be made responsible for the measured positive inotropic effects on frog heart of thrombin.
Abstract: The cortical potential visually evoked by motion of a periodic grating (motion VEP) is composed of a transient component which decays within 500 ms of stimulus-onset (motion-on VEP) and a sustained component. Amplitude and peak latency of wave N2 of the motion-on VEP are functions of grating velocity. Both remain constant at spatial frequencies between 0.6 and 4.3 c/deg and at temporal frequencies within the equivalent intervals. The transient component of the motion VEP is independent of the spatial phase position of the grating before motion onset. The sustained component can only be seen in the averaged motion VEP at constant phase position of the grating before motion onset. This potential consists of periodical fluctuations with a main frequency equal to the temporal frequency of the moving grating. As a result of psychophysical investigations some authors suggest pattern velocity is the relevant variable of velocity perception, others temporal frequency. The motion VEP is dependent on both velocity and temporal frequency, the transient component is a function of velocity, the sustained component of temporal frequency.
Abstract: 1. A single glass micropipette voltage clamp technique with intracellular dialysis was used to study Ba2+ currents in isolated ventricular cells from guinea-pig hearts. Effects of the 1,4-dihydropyridine Bay K 8644 on whole-cell currents were evaluated at 37 degrees C. 2. Bay K 8644 increased the Ba2+ peak currents at test potentials between -50 and +20 mV and shifted the current-voltage relationships towards hyperpolarizing potentials (leftward shift for Ca2+ channel activation, 13.8 +/- 4.1 mV; n = 9; Bay K 8644, 5 mumol/l). 3. The peak times of the Ba2+ currents were diminished over the voltage range tested between -40 and +20 mV after Bay K 8644 in parallel with a shortening of the time constant of activation that was estimated from fits of the recorded currents with a d2f model. 4. The decay of the Ba2+ currents was fitted with two exponentials including a pedestal. The compound Bay K 8644 accelerated the fast decay over the whole voltage range. The amplitude of the rapidly inactivated component of the Ba2+ currents was strikingly increased after application of Bay K 8644. 5. The steady-state inactivation using a 0.5 or 5 s pre-pulse was shifted towards hyperpolarizing potentials (leftward shift 10.3 +/- 5.2 mV; n = 4; Bay K 8644, 5 mumol/l). 6. The change in the time course of Bay K 8644-modified Ba2+ currents cannot be described solely by a decrease of the backward rate coefficient from an open to a closed state of the Ca2+ channel (Sanguinetti, Krafte & Kass, 1986). The described effects of Bay K 8644 on the inactivation can be both qualitatively and quantitatively described by a model of current-dependent inactivation (Standen & Stanfield, 1982), assuming a lower affinity of an internal binding site for Ba2+ than for Ca2+.
Abstract: Effects of the proteolytic enzyme thrombin in the modulation of cardiac Ca-channel currents were examined in single ventricular cells from frog myocardium, using the whole-cell voltage clamp technique (Hamill et al. 1981). Application of 3.8.10(-9) M thrombin to the bath increased the peak of the Ca-channel current by 84 +/- 35% (8 cells). Hirudin (31.10(-9) M), a specific thrombin inhibitor, blocked the thrombin-induced increase of this current. The increase in the current can be made responsible for the measured positive inotropic effects of thrombin as well as for the shift of the plateau voltage of the action potentials towards more positive values.
Abstract: A single glass micropipette voltage clamp technique with intracellular dialysis was used to study the effects of the trapidil derivatives AR 12-456 and AR 12-463 on Ca channel currents carried by Ba2+ in isolated ventricular cells from mice hearts. Inspite of a more potent inhibition of the cAMP phosphodiesterase from heart (Bartel et al. 1985) a reversible Ca channel blocking action of both compounds could be observed. The concentration of half maximal block was calculated to about 50 mumol/l for both derivatives tested. Neither a shift in the current-voltage relationships nor a significant change in the potential for half maximal activation was found. The maximal Ba2+-conductance was reduced. The steady state inactivation was shifted towards more negative potentials by application of 100 mumol/l AR 12-463. The decay of the Ba currents was accelerated in the range of the applied test potentials between -20 and +20 mV. It is concluded that the new trapidil derivatives with more potent inhibitory action on cardiac phosphodiesterase than trapidil can block myocardial Ca channels.
Abstract: Ventricular cells of adult mice were prepared by an enzyme digestion procedure. Single channel currents were recorded by a conventional patch clamp technique from cell attached patches. Voltage steps from the holding potential of -80 mV to test potentials between -35 and +50 mV caused openings of two types of outward currents through single channels with the conductances of 27 and 12 pS, respectively. The averaged currents reveal transient time courses for both channel types. The current-voltage relations of both single channel currents were linear over the tested voltage range and intersected the voltage axis at -70 mV. This indicates that both single channel currents are mainly carried by potassium ions. All open and closed times were found to be voltage independent. The 27 pS channel had a mean open time of 3.9 +/- 1.0 ms (n = 8). The closed time consisted of two components with tau 1 = 2.1 +/- 0.2 ms and tau 2 = 50 +/- 19 ms (n = 8). The 12 pS channel had a mean open time of 34.0 +/- 5.2 ms (n = 3) and the two components of the mean closed time have been calculated as tau 1 = 8.3 +/- 2.1 ms and tau 2 = 120 +/- 50 ms (n = 3; all mean +/- SD).
Abstract: Lidocaine block of single cardiac sodium channels was studied in cell free inside-out patches of ventricular cells isolated from guinea-pig hearts. When applied to the inner surface of the membrane lidocaine depressed Na channel currents by decreasing the probability P of the channels to open measured from the peaks of the averaged currents. In parallel to the decrease in P the relative number of empty sweeps (nulls) was increased. Half maximum block of the activity of single Na channels was observed at 2.9 microM. Lidocaine affected the gating behaviour of Na channels by shortening of the mean open time tau 0 from 0.44 +/- 0.17 (control) to 0.19 +/- 0.13 (5 microM lidocaine, holding potential-120 mV, test potential -60 mV). Five micromolar lidocaine completely suppressed burst-like openings of Na channels and abolished the slow decaying phase of the averaged currents. A shift from - 120 towards - 160 mV exerted relief from the effect of both P and tau 0.
Abstract: Single sodium channel currents were analysed in cell attached patches from single ventricular cells of guinea pig hearts in the presence of a novel cardiotonic compound DPI 201-106. The mean single channel conductance of DPI-treated Na channels was not changed by DPI (20.8 +/- 4 pS, control, 3 patches; 21.3 +/- 1 pS with DPI, 5 mumol/1,3 patches). DPI voltage-dependently prolongs the cardiac sodium channel openings by removal of inactivation at potentials positive to -40 mV. At potentials negative to -40 mV a clustering of short openings at the very beginning of the depolarizing voltage steps can be observed causing a transient time course of the averaged currents. Long openings induced an extremely slow inactivation. Short openings, long openings and nulls appeared in groups referring to a modal gating behaviour of DPI-treated sodium channels. DPI-modified Na channels showed a monotonously prolonged mean open time with increased depolarizing voltage steps, e.g. the open state probability within a sweep was increased. However, the number of non-empty sweeps was decreased with the magnitude of the depolarizing steps, e.g. the probability of the channel being open as calculated from the averaged currents was voltage-dependently decreased by DPI (50% decrease at -50.7 +/- 9 9 mV, 3 patches). Short and long openings of DPI-modified channels could be separated by variation of the holding potential. The occurrence of long Na channel openings was much more suppressed by reducing the holding potential (half maximum inactivation at -112 +/- 8 mV, 4 patches) than that of short openings (half maximum inactivation at -88 +/- 8 mV, 4 patches). Otherwise, short living openings completely disappeared at potentials positive to -40 mV where the occurrence of long openings was favoured. The differential voltage dependence of blocking and activating effects of DPI on cardiac Na channels as well as the differential voltage dependence of the appearance of short and long openings refers to a modal gating behaviour of cardiac Na channels.
Abstract: Currents through single Na channels of ventricular cells from adult mouse and guinea pig hearts were studied using the patch clamp technique. Under control conditions the majority of openings is brief and a clustering at the beginning of the depolarizing pulse can be observed. Only between 1 and 8% of the sweeps show long lasting bursts. The bursting may account for a second slow phase of decay of macroscopic currents. In the presence of aconitine in the pipette up to 80% of the sweeps showed bursts underlying the slow inactivation of aconitine modified macroscopic currents. The mean open times are unchanged but the number of openings per sweep is dramatically increased due to aconitine. It is discussed that Na channels may function in different "modes". One mode is characterized by a fast transition into an absorbing state the other one by frequent reopenings ("bursts"). aconitine favours the "bursting" mode.
Abstract: Our intention was to obtain a visual evoked potential (VEP) consisting only of a movement-related component for the purpose of further investigations of movement detection. This was attempted by selection of appropriate stimulus conditions. Evoked by initial movement a VEP with five typical waves was observed at the human occipital scalp. The N2-wave with a peak latency of 180-200 ms was most prominent. Following results were yielded in the experiments carried out: 1. Adaptation to a pattern movement: The amplitude of N2 and P1 is significantly reduced (Fig. 4). 2. Relation between amplitude and velocity: The experimental data could be approximated by a power function with an exponent of m = 0.3 for N2 and lower m for later waves (Fig. 5). 3. Pattern variation (grating, checkerboard, zig-zag) had no influence on N2 but on P1 and possibly also on later waves (Fig. 6). These results suggest that the wave N2 is movement-related under our experimental conditions. A pattern-related component may additionally be assumed in wave P2. Components, evoked by further reasons, may be included in the waves following N2. Their specification demands supplementary experiments.
