Characterization of inositol-1,4,5-trisphosphate-gated channels in the plasma membrane of rat olfactory neurons. (33/18215)

It is generally accepted that inositol-1,4,5-trisphosphate (InsP3) plays a role in olfactory transduction. However, the precise mode of action of InsP3 remains controversial. We have characterized the conductances activated by the addition of 10 microM InsP3 to excised patches of soma plasma membrane from rat olfactory neurons. InsP3 induced current fluctuations in 25 of 121 inside-out patches. These conductances could be classified into two groups according to the polarity of the current at a holding potential of +40 to +60 mV (with Ringer's in the pipette and pseudointracellular solution in the bath). Conductances mediating outward currents could be further divided into large- (64 +/- 4 pS, n = 4) and small- (16 +/- 1.7 pS, n = 11) conductance channels. Both small- and large-conductance channels were nonspecific cation channels. The large-conductance channel displayed bursting behavior at +40 mV, with flickering increasing at negative holding potentials to the point where single-channel currents were no longer discernible. The small-conductance channel did not display flickering behavior. The conductance mediating inward currents at +40 to +60 mV reversed at +73 +/- 4 mV (n = 4). The current traces displayed considerable fluctuations, and single-channel currents could not be discerned. The current fluctuations returned to baseline after removal of InsP3. The power density spectrum for the excess noise generated by InsP3 followed a 1/f dependence consistent with conductance fluctuations in the channel mediating this current, although other mechanisms are not excluded. These experiments demonstrate the presence of plasma membrane InsP3-gated channels of different ionic specificity in olfactory receptor cells.  (+info)

Modulation of the bursting properties of single mouse pancreatic beta-cells by artificial conductances. (34/18215)

Glucose triggers bursting activity in pancreatic islets, which mediates the Ca2+ uptake that triggers insulin secretion. Aside from the channel mechanism responsible for bursting, which remains unsettled, it is not clear whether bursting is an endogenous property of individual beta-cells or requires an electrically coupled islet. While many workers report stochastic firing or quasibursting in single cells, a few reports describe single-cell bursts much longer (minutes) than those of islets (15-60 s). We studied the behavior of single cells systematically to help resolve this issue. Perforated patch recordings were made from single mouse beta-cells or hamster insulinoma tumor cells in current clamp at 30-35 degrees C, using standard K+-rich pipette solution and external solutions containing 11.1 mM glucose. Dynamic clamp was used to apply artificial KATP and Ca2+ channel conductances to cells in current clamp to assess the role of Ca2+ and KATP channels in single cell firing. The electrical activity we observed in mouse beta-cells was heterogeneous, with three basic patterns encountered: 1) repetitive fast spiking; 2) fast spikes superimposed on brief (<5 s) plateaus; or 3) periodic plateaus of longer duration (10-20 s) with small spikes. Pattern 2 was most similar to islet bursting but was significantly faster. Burst plateaus lasting on the order of minutes were only observed when recordings were made from cell clusters. Adding gCa to cells increased the depolarizing drive of bursting and lengthened the plateaus, whereas adding gKATP hyperpolarized the cells and lengthened the silent phases. Adding gCa and gKATP together did not cancel out their individual effects but could induce robust bursts that resembled those of islets, and with increased period. These added currents had no slow components, indicating that the mechanisms of physiological bursting are likely to be endogenous to single beta-cells. It is unlikely that the fast bursting (class 2) was due to oscillations in gKATP because it persisted in 100 microM tolbutamide. The ability of small exogenous currents to modify beta-cell firing patterns supports the hypothesis that single cells contain the necessary mechanisms for bursting but often fail to exhibit this behavior because of heterogeneity of cell parameters.  (+info)

Electrically excitable normal rat kidney fibroblasts: A new model system for cell-semiconductor hybrids. (35/18215)

In testing various designs of cell-semiconductor hybrids, the choice of a suitable type of electrically excitable cell is crucial. Here normal rat kidney (NRK) fibroblasts are presented as a cell line, easily maintained in culture, that may substitute for heart or nerve cells in many experiments. Like heart muscle cells, NRK fibroblasts form electrically coupled confluent cell layers, in which propagating action potentials are spontaneously generated. These, however, are not associated with mechanical disturbances. Here we compare heart muscle cells and NRK fibroblasts with respect to action potential waveform, morphology, and substrate adhesion profile, using the whole-cell variant of the patch-clamp technique, atomic force microscopy (AFM), and reflection interference contrast microscopy (RICM), respectively. Our results clearly demonstrate that NRK fibroblasts should provide a highly suitable test system for investigating the signal transfer between electrically excitable cells and extracellular detectors, available at a minimum cost and effort for the experimenters.  (+info)

Identification of mechanosensitive ion channels in the cytoplasmic membrane of Corynebacterium glutamicum. (36/18215)

Patch-clamp experiments performed on membrane fragments of Corynebacterium glutamicum fused into giant liposomes revealed the presence of two different stretch-activated conductances, 600 to 700 pS and 1,200 to 1,400 pS in 0.1 M KCl, that exhibited the same characteristics in terms of kinetics, ion selectivity, and voltage dependence.  (+info)

Confocal calcium imaging reveals an ionotropic P2 nucleotide receptor in the paranodal membrane of rat Schwann cells. (37/18215)

1. The paranodal Schwann cell region is of major importance for the function of a myelinated axon. In the present study we searched for a possible ionotropic effect of extracellular ATP in this Schwann cell compartment. 2. Whole-cell patch-clamp recordings from cultured rat Schwann cells revealed that ATP and 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP) induced a non-specific cation current. The effect of ATP was much enhanced in a Ca2+- and Mg2+-free solution. ADP, UTP and alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP) had no effect. 3. Confocal Ca2+ imaging of myelinating Schwann cells in isolated rat spinal roots showed a BzATP-induced rise in the free intracellular Ca2+ concentration in the paranodal Schwann cell cytoplasm whereas alpha,beta-meATP and 2-(methylthio)-adenosine 5'-triphosphate were without effect. In contrast to the known metabotropic effect of UTP on these Schwann cell regions, the BzATP-induced Ca2+ signal was not transient, was unaffected by depletion of intracellular Ca2+ stores and dependent on the presence of extracellular Ca2+. 4. These results suggest that an ionotropic ATP receptor with electrophysiological and pharmacological characteristics of the P2X7 subtype of nucleotide receptors is functionally active in myelinating Schwann cells of peripheral nerves. Such a receptor might contribute to Schwann cell reactions in nerve injury or neuropathy.  (+info)

Depolarization-evoked Ca2+ release in a non-excitable cell, the rat megakaryocyte. (38/18215)

1. The effect of membrane potential on [Ca2+]i in rat megakaryocytes was studied using simultaneous whole-cell patch clamp and fura-2 fluorescence recordings. 2. Depolarization from -75 to 0 mV had no effect on [Ca2+]i in unstimulated cells, but evoked one or more spikes of Ca2+ increase (peak increase: 714 +/- 95 nM) during activation of metabotropic purinoceptors by 1 microM ADP. 3. The depolarization-evoked Ca2+ increase was present in Ca2+-free medium and also following removal of Na+. Thus depolarization mobilizes Ca2+ from an intracellular store without a requirement for altered Na+-Ca2+ exchange activity. 4. Intracellular dialysis with heparin blocked the depolarization-evoked Ca2+ increase, indicating a role for functional IP3 receptors. 5. Under current clamp, ADP caused the membrane potential to fluctuate between -43 +/- 1 and -76 +/- 1 mV. Under voltage clamp, depolarization from -75 to -45 mV evoked a transient [Ca2+]i increase (398 +/- 91 nM) during exposure to ADP. 6. We conclude that during stimulation of metabotropic purinoceptors, membrane depolarization over the physiological range can stimulate Ca2+ release from intracellular stores in the rat megakaryocyte, a non-excitable cell type. This may represent an important mechanism by which electrogenic influences can control patterns of [Ca2+]i increase.  (+info)

A repetitive mode of activation of discrete Ca2+ release events (Ca2+ sparks) in frog skeletal muscle fibres. (39/18215)

1. Ca2+ release events (Ca2+ 'sparks'), which are believed to arise from the opening of a sarcoplasmic reticulum (SR) Ca2+ release channel or a small cluster of such channels that act as a release unit, have been measured in single, frog (Rana pipiens) skeletal muscle fibres. 2. Under conditions of extremely low rates of occurrence of Ca2+ sparks we observed, within individual identified triads, repetitive Ca2+ release events which occurred at a frequency more than 100-fold greater than the prevailing average event rate. Repetitive sparks were recorded during voltage-clamp test depolarizations after a brief (0.3-2 s) repriming interval in fibres held at 0 mV and in chronically depolarized, 'notched' fibres. 3. These repetitive events are likely to arise from the re-opening of the same SR Ca2+ release channel or release unit operating in a repetitive gating mode ('rep-mode'), rather than from the random activation of multiple, independent channels or release units within a triad. A train of rep-mode events thus represents a series of Ca2+ sparks arising from a single location within the fibre. Rep-mode events are activated among different triads in a random manner after brief repriming. The frequency of repetitive events among all identified events during voltage-clamp depolarization to 0 mV after brief repriming was 3.9 +/- 1.3 %. The occurrence of repetitive events was not related to exposure of the fibre to laser illumination. 4. The events observed within a rep-mode train exhibited a relatively uniform amplitude. Analysis of intervals between identified events in triads exhibiting rep-mode trains indicated similar variations of fluorescence as in neighbouring, quiescent triads, suggesting there was not a significant number of small, unidentified events at the triads exhibiting rep-mode activity. 5. The distribution of rep-mode interspark intervals exhibited a paucity of events at short intervals, consistent with the need for recovery from inactivation before activation of the next event in a repetitive train. The mean interspark interval of repetitive sparks during voltage-clamp depolarizations was 88 +/- 5 ms, and was independent of membrane potential. 6. The individual Ca2+ sparks within a rep-mode train were similar in average amplitude and spatiotemporal extent to singly occurring sparks, suggesting a common mechanism for termination of the channel opening(s) underlying both types of events. The average properties of the sparks did not vary during a train. The relative amplitude of a spark within a rep-mode was not correlated with its rise time. 7. Repetitive Ca2+ release events represent a mode of gating of SR Ca2+ release channels which may be significant during long depolarizations and which may be influenced by the biochemical state of the SR ryanodine receptor Ca2+ release channels.  (+info)

Glucose-receptive neurones in the rat ventromedial hypothalamus express KATP channels composed of Kir6.1 and SUR1 subunits. (40/18215)

1. Patch-clamp recordings were made from rat ventromedial hypothalamic neurones in slices of brain tissue in vitro. In cell-attached recordings, removal of extracellular glucose or metabolic inhibition with sodium azide reduced the firing rate of a subpopulation of cells through the activation of a 65 pS channel that was blocked by the sulphonylureas tolbutamide and glibenclamide. 2. In whole-cell patch-clamp recordings, in the absence of ATP in the electrode solution, glucose-receptive neurones gradually hyperpolarized due to the induction of an outward current at -60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide. 3. In recordings where the electrode solution contained 4 mM ATP, this outward current was not observed. Under these conditions, 500 microM diazoxide was found to induce an outward current that was blocked by tolbutamide. 4. In cell-attached recordings diazoxide and the active fragment of leptin (leptin 22-56) reduced the firing rate of glucose-receptive neurones by the activation of a channel with similar properties to that induced by removal of extracellular glucose. 5. Reverse transcription followed by the polymerase chain reaction using cytoplasm from single glucose-receptive neurones demonstrated the expression of the ATP-sensitive potassium (KATP) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2. 6. It is concluded that glucose-receptive neurones within the rat ventromedial hypothalamus exhibit a KATP channel current with pharmacological and molecular properties similar to those reported in other tissues.  (+info)