CaM kinase augments cardiac L-type Ca2+ current: a cellular mechanism for long Q-T arrhythmias.
(25/1812)
Early afterdepolarizations (EAD) caused by L-type Ca2+ current (ICa, L) are thought to initiate long Q-T arrhythmias, but the role of intracellular Ca2+ in these arrhythmias is controversial. Rabbit ventricular myocytes were stimulated with a prolonged EAD-containing action potential-clamp waveform to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) in ICa,L during repolarization. ICa,L was initially augmented, and augmentation was dependent on Ca2+ from the sarcoplasmic reticulum because the augmentation was prevented by ryanodine or thapsigargin. ICa,L augmentation was also dependent on CaM kinase, because it was prevented by dialysis with the inhibitor peptide AC3-I and reconstituted by exogenous constitutively active CaM kinase when Ba2+ was substituted for bath Ca2+. Ultrastructural studies confirmed that endogenous CaM kinase, L-type Ca2+ channels, and ryanodine receptors colocalized near T tubules. EAD induction was significantly reduced in current-clamped cells dialyzed with AC3-I (4/15) compared with cells dialyzed with an inactive control peptide (11/15, P = 0.013). These findings support the hypothesis that EADs are facilitated by CaM kinase. (+info)
Effect of extracellular cations on the inward rectifying K+ channels Kir2.1 and Kir3.1/Kir3.4.
(26/1812)
The effects of Ba2+, Mg2+, Ca2+ and Na+ as blocking ions were investigated in 90 and 10 mM extracellular K+ solutions on the cloned inward rectifying K+ channel Kir2.1 expressed in Xenopus oocytes. Some data were also obtained using another inward rectifying K+ channel Kir3.1/Kir3.4. The addition of Ba2+ caused a concentration-, voltage- and time-dependent block of both channels. Decreasing the extracellular K+ concentration augmented the block. The data suggest that Ba2+ blocks the channels by binding to a site within the channel pore and that the electrical binding distance, delta, of the site is significantly different for Kir2.1 and Kir3. 1/Kir3.4 (0.38 and 0.22, respectively). Mg2+ and Ca2+ caused an instantaneous concentration- and voltage-dependent block of both channels. With Kir2.1, decreasing the K+ concentration augmented the block. The voltage dependence of the block was less than that of Ba2+ ([delta], 0.1), indicating a more superficial binding site for these ions within the channel pore. The affinity of the channels for Mg2+ and Ca2+ was 1000-fold lower than that for Ba2+. Addition of Na+ resulted in a concentration-, voltage- and time-dependent block of Kir2.1, similar to that observed with Ba2+. The competition between the blocking cations (for Kir2.1: Ba2+, Mg2+, Ca2+; for Kir3. 1/Kir3.4: Ba2+) and extracellular K+ suggests that the binding sites for the blocking cations may be sites to which K+ binds as part of the normal passage of K+ through the channels. It is possible that under normal physiological conditions naturally occurring extracellular cations may partly block the two inward rectifying K+ channels. (+info)
Developmental changes in low and high voltage-activated calcium currents in acutely isolated mouse vestibular neurons.
(27/1812)
1. The development of low voltage-activated (LVA) and high voltage-activated (HVA) calcium currents was studied in neurons acutely dissociated from mouse vestibular ganglia at embryonic stages (E)14, 15, 17 and birth using the whole-cell patch-clamp technique. 2. LVA current was present in almost all neurons tested at stages E14 to E17, although at birth this current was restricted to a few neurons. Two populations of neurons were characterized based on the amplitude of the LVA current. In the first population, LVA current densities decreased between E17 and birth by which time this current tended to disappear in most neurons. A second population of neurons with high density LVA current appeared at E17, and in this group the mean density increased during development. 3. Among HVA currents, the dihydropyridine-sensitive L-type current remained constant between E15 and birth. Over the same period, the density of N- and Q-type currents continuously increased as shown using omega-conotoxin-GVIA (N-type), and high concentrations of omega-agatoxin-IVA (Q-type). The P-type current, sensitive to low concentrations of omega-agatoxin-IVA, transiently increased between E15 and E17, and then both current density and its proportion of the global current decreased. 4. Our results reveal large modifications in the expression of voltage-dependent calcium channels during embryonic development of primary vestibular neurons. The changes in the expression of LVA current and the transient augmentation of P-type HVA current occur during a period characterized by massive neuronal growth and by the beginning of synaptogenesis. These results suggest a specific role of these currents in the ontogenesis of vestibular primary afferents. (+info)
1. The membrane potential of gastrointestinal smooth muscles determines the open probability of ion channels involved in rhythmic electrical activity. The role of Ba2+-sensitive K+ conductances in the maintenance of membrane potential was examined in canine proximal colon circular muscle. 2. Application of Ba2+ (1-100 microM) to strips of tunica muscularis produced depolarization of cells along the submucosal surface of the circular muscle layer. Significantly higher concentrations of Ba2+ were needed to depolarize preparations from which the submucosal and myenteric pacemaker regions were removed. 3. Elevation of extracellular [K+]o (from 5.9 to 12 mM) brought membrane potentials closer to EK (the Nernst potential for K+ ions), suggesting activation of a K+ conductance. This occurred at potentials much more negative than the activation range for delayed rectifier channels (Kv). 4. Forskolin (1 microM) caused hyperpolarization and a leftward shift in the dose-response relationship for Ba2+, suggesting that forskolin may activate a Ba2+-sensitive conductance. 5. Patch-clamp recordings from interstitial cells of Cajal (ICC) revealed the presence of a Ba2+-sensitive inward rectifier potassium conductance. Far less of this conductance was present in smooth muscle cells. 6. Kir2.1 was expressed in the circular muscle layer of the canine proximal colon, duodenum, jejunum and ileum. Kir2.1 mRNA was expressed in greater abundance along the submucosal surface of the circular muscle layer in the colon. 7. These results demonstrate that ICC express a Ba2+-sensitive conductance (possibly encoded by Kir2.1). This conductance contributes to the generation and maintenance of negative membrane potentials between slow waves. (+info)
Stimulation of Drosophila TrpL by capacitative Ca2+ entry.
(29/1812)
Trp-like protein (TrpL, where Trp is transient receptor-potential protein) of Drosophila, a non-selective cation channel activated in photoreceptor cells by a phospholipase C-dependent mechanism, is thought to be a prototypical receptor-activated channel. Our previous studies showed that TrpL channels are not activated by depletion of internal Ca2+ stores when expressed in Sf9 cells. Using fura-2 to measure cation influx via TrpL, and cell-attached patch recordings to monitor TrpL single-channel activity directly, we have found a thapsigargin-induced increase in TrpL activity in the presence of extracellular bivalent cations, with Ca2+>Sr2+>> Ba2+. The increase in TrpL channel activity was blocked by concentrations of La3+ that completely inhibited endogenous capacitative Ca2+ entry (CCE), but have no effect on TrpL, suggesting that TrpL exhibits trans-stimulation by cation entry via CCE. TrpL has two putative calmodulin (CaM)-binding domains, designated CBS-1 and CBS-2. To determine which site may be required for stimulation of TrpL by the cytosolic free Ca2+ concentration ([Ca2+]i), a chimaeric construct was created in which the C-terminal domain of TrpL containing CBS-2 was attached to human TrpC1, a short homologue of Trp that is not activated by depletion of internal Ca2+ stores or by a rise in [Ca2+]i. This gain-of-function mutant, designated TrpC1-TrpL, exhibited trans-stimulation by Ca2+ entry via CCE. Examination of CaM binding in gel-overlay experiments showed that TrpL and the TrpC1-TrpL chimaera bound CaM, but TrpC1 or a truncated version of TrpL lacking CBS-2 did not. These results suggest that only CBS-2 binds CaM in native TrpL and that the C-terminal domain containing this site is important for trans-stimulation of TrpL by CCE. (+info)
The relationship between distal tubular proton secretion and dietary potassium depletion: evidence for up-regulation of H+ -ATPase.
(30/1812)
BACKGROUND: Dietary potassium depletion is associated with elevated plasma bicarbonate concentration and enhanced bicarbonate reabsorption in the distal tubule. The relationship between distal proton secretion and potassium status was investigated by in vivo microperfusion of the superficial distal tubule. METHODS: Experiments were performed on anaesthetized rats that had been maintained on either a low-potassium or control diet for 3-5 weeks prior to experimentation. The distal tubules were perfused at 10 nl/min with either a standard or a barium chloride-containing solution, and the late distal tubular transepithelial potential difference (Vte) and pH of the luminal fluid were recorded using a double-barrelled voltage and ion-sensitive microelectrode. RESULTS: In control rats, the Vte was -40.7+/-2.4 mV and the tubular fluid pH was 6.44+/-0.07; in potassium-depleted animals, the Vte was -15.0+/-1.4 mV and the pH was 6.76+/-0.03. The pH values in both groups of animals were significantly lower than would be predicted from the Vte and systemic pH for passive H+ distribution, indicating active proton secretion. Moreover, in hypokalaemic rats, this difference from predicted pH was significantly greater than in control animals (control = 0.27+/-0.06 vs. low-potassium = 0.46+/-0.03; P<0.01), suggesting enhanced active proton secretion. During perfusion with a solution containing BaCl2, the late distal tubule Vte became lumen positive in potassium-depleted rats, contrasting with an increased lumen negativity in potassium-replete controls. The barium-induced lumen-positive potential difference observed in the hypokalaemic rats was abolished by intravenous administration of acetazolamide. CONCLUSION: These data are consistent with enhanced electrogenic proton secretion (H+ -ATPase) during dietary potassium deprivation. (+info)
Evidence for chloride ions as intracellular messenger substances in astrocytes.
(31/1812)
Cultured rat hippocampal astrocytes were used to investigate the mechanism underlying the suppression of Ba2+-sensitive K+ currents by GABAA receptor activation. Muscimol application had two effects on whole cell currents: opening of the well-known Cl- channel of the GABAA receptor and a secondary longer-lasting blockade of outward K+ currents displaying both peak and plateau phases. This blockade was independent of both Na+ (inside and outside) and ATP in the pipette. It also seemed to be independent of muscimol binding to the receptor because picrotoxin application showed no effect on the K+ conductance. The effect is blocked when anion efflux is prevented by replacing Cl- with gluconate (both inside and out) and is enhanced with more permeant anions such as Br- and I-. Moreover, the effect is reproduced in the absence of muscimol by promoting Cl- efflux via lowering of extracellular Cl- levels. These results, along with the requirement for Cl- efflux in muscimol experiments, show a strong dependency of the secondary blockade on Cl- efflux through the Cl- channel of the GABAA receptor. We therefore conclude that changes in the intracellular Cl- concentration alter the outward K+ conductances of astrocytes. Such a Cl--mediated modulation of an astrocytic K+ conductance will have important consequences for the progression of spreading depression through brain tissue and for astrocytic swelling in pathological situations. (+info)
Muscarinic receptor activity has multiple effects on the resting membrane potentials of CA1 hippocampal interneurons.
(32/1812)
Inhibitory interneurons appear to be an important target for the muscarinic actions of cholinergic inputs to the hippocampus. We investigated the effect of muscarinic receptor activity on the membrane potential (V(m)) and currents of rat hippocampal CA1 interneurons using whole-cell recording from visually identified CA1 interneurons. The predominant response observed was a muscarinic depolarization that was detected in interneurons from all layers of CA1. This depolarization was mediated by at least two mechanisms: a reduction in a potassium current and a mechanism that depended on extracellular sodium. Other interneurons responded to muscarinic agonists with a hyperpolarization or a biphasic response (hyperpolarization followed by depolarization). Hyperpolarizations and biphasic responses were found in all layers of CA1 but more frequently in stratum radiatum and stratum lacunosum moleculare. Muscarinic hyperpolarization was caused by the activation of a barium- and cesium-sensitive inwardly rectifying potassium channel. A small number of interneurons, primarily in or bordering the stratum pyramidale, produced slow membrane potential (0.04 Hz) oscillations. Many interneurons did not respond to muscarinic activity at all; half of these were in the stratum oriens. There was no strong correlation between any changes in V(m) response to muscarine and morphology, as determined by reconstruction of the interneurons. It was not possible to predict the morphology or the layer distribution of an interneuron based on the type of muscarinic membrane potential response it had. This lack of correlation between muscarinic function and morphology implies a greater complexity of interneuron function than has been realized previously. (+info)