Hypo-osmotic potentiation of acetylcholine-stimulated ciliary beat frequency through ATP release in rat tracheal ciliary cells

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video-enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. An adequate hypo-osmotic stress (−40 mos m ) potentiated ACh-stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose–response curve to the left (lower concentration side). This potentiation was independent of hypo-osmotic stresses applied ranging from −20 mosM to −90 mosM. A hypo-osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo-osmotic potentiation of ACh-stimulated CBF increase and that ATP increased [Ca²⁺]_i and CBF, as well as potentiating ACh-stimulated rises in [Ca²⁺]_i and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X₄ purinergic receptor. A hypo-osmotic stress (−40 mosM) transiently increased [Ca²⁺]_i and potentiated the ACh-stimulated [Ca²⁺]_i increase. The hypo-osmotic potentiation of ACh-stimulated CBF increase was not detected under Ca²⁺-free conditions. These observations suggest that a hypo-osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca²⁺]_i and CBF in ACh-stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X₄.     

Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells

Treatment of human epithelial kidney (HEK293) cells with low concentrations of the muscarinic agonist methacholine results in the activation of complex and repetitive cycling of intracellular calcium ([Ca²⁺]_i), known as [Ca²⁺]_i oscillations. These oscillations occur with a frequency that depends on the concentration of methacholine, whereas the magnitude of the [Ca²⁺]_i spikes does not. The oscillations do not persist in the absence of extracellular Ca²⁺, leading to the conclusion that entry of Ca²⁺ across the plasma membrane plays a significant role in either their initiation or maintenance. However, treatment of cells with high concentrations of GdCl₃, a condition which limits the flux of calcium ions across the plasma membrane in both directions, allows sustained [Ca²⁺]_i oscillations to occur. This suggests that the mechanisms that both initiate and regenerate [Ca²⁺]_i oscillations are intrinsic to the intracellular milieu and do not require entry of extracellular Ca²⁺. This would additionally suggest that, under normal conditions, the role of calcium entry is to sustain [Ca²⁺]_i oscillations. By utilizing relatively specific pharmacological manoeuvres we provide evidence that the Ca²⁺ entry that supports Ca²⁺ oscillations occurs through the store-operated or capacitative calcium entry pathway. However, by artificial introduction of a non-store-operated pathway into the cells (TRPC3 channels), we find that other Ca²⁺ entry mechanisms can influence oscillation frequency in addition to the store-operated channels.     

Mechanisms underlying variations in excitation–contraction coupling across the mouse left ventricular free wall

Ca²⁺ release during excitation–contraction (EC) coupling varies across the left ventricular free wall. Here, we investigated the mechanisms underlying EC coupling differences between mouse left ventricular epicardial (Epi) and endocardial (Endo) myocytes. We found that diastolic and systolic [Ca²⁺]_i was higher in paced Endo than in Epi myocytes. Our data indicated that differences in action potential (AP) waveform between Epi and Endo cells only partially accounted for differences in [Ca²⁺]_i. Rather, we found that the amplitude of the [Ca²⁺]_i transient, but not its trigger – the Ca²⁺ current – was larger in Endo than in Epi cells. We also found that spontaneous Ca²⁺ spark activity was about 2.8-fold higher in Endo than in Epi cells. Interestingly, ryanodine receptor type 2 (RyR2) protein expression was nearly 2-fold higher in Endo than in Epi myocytes. Finally, we observed less Na⁺–Ca²⁺ exchanger function in Endo than in Epi cells, which was associated with decreased Ca²⁺ efflux during the AP; this contributed to higher diastolic [Ca²⁺]_i and SR Ca²⁺ in Endo than in Epi cells during pacing. We propose that transmural differences in AP waveform, SR Ca²⁺ release, and Na⁺–Ca²⁺ exchanger function underlie differences in [Ca²⁺]_i and EC coupling across the left ventricular free wall.     

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.     

Electrophysiological properties of BK channels in Xenopus motor nerve terminals

Single channel properties of Ca²⁺-activated K⁺ (BK or Maxi-K) channels have been investigated in presynaptic membranes in Xenopus motoneurone–muscle cell cultures. The occurrence and density of BK channels increased with maturation/synaptogenesis and was not uniform: highest at the release face of bouton-like synaptic varicosities in contact with muscle cells, and lowest in varicosities that did not contact muscle cells. The Ca²⁺ affinity of the channel ( K _d= 7.7 μ m at a membrane potential of +20 mV) was lower than those of BK channels that have been characterized in other terminals. Hill coefficients varied between 1.5 and 2.8 at different potentials and open probability increased e-fold per 16 mV change in membrane potential over a range of [Ca²⁺]_i from 1 μ m to 1 m m . The maximal activation rate of ensembled single BK channel currents was in the submillisecond range at ≥+20 mV. The activation rate increased ∼10-fold in response to a [Ca²⁺]_i increase from 1 to 100 μ m , but increased only ∼2-fold with a voltage change from +20 to +130 mV. The fastest activation kinetics of BK channels in cell-attached patches resembled that in inside-out patches with [Ca²⁺]_i of 100 μ m or more, suggesting that many BK channels are located very close to calcium channels. Given the low Ca²⁺ affinity and rapid Ca²⁺ binding/unbinding properties, we conclude that BK channels in this preparation are adapted to play an important role in regulation of neurotransmitter release, and they are ideal reporters of local [Ca²⁺] at the inner membrane surface.     

Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca2+]i and Cl− channels

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca²⁺]_i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca²⁺]_i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca²⁺]_i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca²⁺]_i were abolished by loading the preparations with the Ca²⁺ chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP₃ induced Ca²⁺ release, by N -ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl⁻)-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP₃ formation. This causes Ca²⁺ release from intracellular stores which activates Ca²⁺-dependent Cl⁻ channels.     

Role of the store-operated calcium entry proteins Stim1 and Orai1 in muscarinic cholinergic receptor-stimulated calcium oscillations in human embryonic kidney cells

We have investigated the nature of the Ca²⁺ entry supporting [Ca²⁺]_i oscillations in human embryonic kidney (HEK293) cells by examining the roles of recently described store-operated Ca²⁺ entry proteins, Stim1 and Orai1. Knockdown of Stim1 by RNA interference (RNAi) reduced the frequency of [Ca²⁺]_i oscillations in response to a low concentration of methacholine to the level seen in the absence of external Ca²⁺. However, knockdown of Stim1 did not block oscillations in canomical transient receptor potential 3 channel (TRPC3)-expressing cells and did not affect Ca²⁺ entry in response to arachidonic acid. The effects of knockdown of Stim1 could be reversed by inhibiting Ca²⁺ extrusion with a high concentration of Gd³⁺, or by rescuing the knockdown by overexpression of Stim1. Similarly, knockdown of Orai1 abrogated [Ca²⁺]_i oscillations, and this was reversed by use of high concentrations of Gd³⁺; however, knockdown of Orai1 did not affect arachidonic acid-activated entry. RNAi targeting 34 members of the transient receptor potential (TRP) channel superfamily did not reveal a role for any of these channel proteins in store-operated Ca²⁺ entry in HEK293 cells. These findings indicate that the Ca²⁺ entry supporting [Ca²⁺]_i oscillations in HEK293 cells depends upon the Ca²⁺ sensor, Stim1, and calcium release-activated Ca²⁺ channel protein, Orai1, and provide further support for our conclusion that it is the store-operated mechanism that plays the major role in this pathway.     

Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones

Experiments were done using guinea-pig sympathetic neurones dissociated from the stellate ganglia to establish whether calcium-induced calcium release (CICR) modulated action potential (AP) generation in mammalian neurones. Using measurements of intracellular calcium ([Ca²⁺]_i) with the Ca²⁺-sensitive dye fluo-3, we demonstrated that 10 m m caffeine activated ryanodine receptors and caused a rise in [Ca²⁺]_i in both Ca²⁺-containing and Ca²⁺-deficient solutions. We also demonstrated that combined treatment with caffeine and 1 μ m thapsigargin or caffeine and 20 μ m ryanodine blocked subsequent caffeine-induced elevations of [Ca²⁺]_i. Treatment with thapsigargin, ryanodine or 200 μ m Cd²⁺ to disrupt CICR decreased the latency to AP generation during 400 ms depolarizing current ramps using the perforated patch whole cell patch clamp in current clamp mode. Treatment with 500 μ m tetraethylammonium also decreased the latency to AP generation during depolarizing current ramps in control cells, but not in cells pretreated with thapsigargin to deplete internal Ca²⁺ stores. In summary, we propose that an outward current, carried at least in part through BK channels, is activated by CICR at membrane voltages approaching the threshold for AP initiation and that this current opposed depolarizing current ramps applied to guinea-pig sympathetic stellate neurones.     

Microheterogeneity of calcium signalling in dendrites

Transient changes in the intracellular concentration of free Ca²⁺ ([Ca²⁺]_i) originating from voltage- or ligand-gated influx and by ligand- or Ca²⁺-gated release from intracellular stores, trigger or modulate many fundamental neuronal processes, including neurotransmitter release and synaptic plasticity. Of the intracellular compartments involved in Ca²⁺ clearance, the endoplasmic reticulum (ER) has received the most attention because it expresses Ca²⁺ pumps and Ca²⁺ channels, thus endowing it with the potential to act as both an intracellular calcium sink and store. We review here our ongoing work on the role of calcium sequestration into, and release from, ER cisterns and the role that this plays in the generation and termination of free [Ca²⁺]_i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. These studies have been approached by combining parallel microfluorometric measurements of free cytosolic [Ca²⁺]_i transients with energy-dispersive X-ray microanalytical measurements of total Ca content within specific dendritic compartments at the electron microscopy level. Our observations support the emerging realization that specific subsets of dendritic ER cisterns provide spatial and temporal microheterogeneity of Ca²⁺ signalling, acting not only as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca²⁺ influx, but also as an intracellular Ca²⁺ source that can be mobilized by a signal cascade originating at activated synapses.     

Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+

At the snake neuromuscular junction, low temperature (LT, 5–7°C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or 'delayed' until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca²⁺]_i directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca²⁺]_i. With brief stimulation at LT, [Ca²⁺]_i transiently increased but returned to baseline (∼63 n m ) in < 8 min. After 15 min at LT, [Ca²⁺]_i was altered by incubating preparations in the Ca²⁺ ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca²⁺]_i increased; the rate at 300 n m Ca²⁺ was ∼double that under basal conditions. Thus the rate of CME – isolated from stimulation, transmitter release, and other forms of endocytosis – is directly influenced by intraterminal Ca²⁺.     

Streptomycin and intracellular calcium modulate the response of single guinea-pig ventricular myocytes to axial stretch

We tested the hypothesis that both stretch-activated channels (SACs) and intracellular calcium ([Ca²⁺]_i) are important in the electrical response of single guinea-pig ventricular myocytes to axial stretch. Myocytes were attached to carbon fibre transducers and stretched, sarcomere length increased by approximately 9 %, and there was a prolongation of the action potential duration. Streptomycin, a blocker of SACs, had no effect upon the shortening, [Ca²⁺]_i transients or action potentials of electrically stimulated, unstretched myocytes, at a concentration of 50 μ m , but at 40 μ m , prevented any stretch-induced increase in action potential duration. Under action potential clamp, stretch elicited a current with a linear current-voltage relationship that was inward at membrane potentials negative to its reversal potential of −30 mV, in 10 of 24 cells tested, and was consistent with the activation of non-specific, cationic SACs. This current was not seen in any stretched cells that were exposed to 40 μ m streptomycin. However, exposure of cells to 5 μ m BAPTA-AM, in order to reduce [Ca²⁺]_i transients, also abolished stretch-induced prolongation of the action potential. We conclude that both SACs and [Ca²⁺]_i are important in the electrical response of cardiac myocytes to stretch, and propose that stretch-induced changes in electrical activity and [Ca²⁺]_i may be linked by inter-dependent mechanisms.     

Simultaneous imaging of Ca2+ signals in interstitial cells of Cajal and longitudinal smooth muscle cells during rhythmic activity in mouse ileum

Electrical rhythmicity in smooth muscle cells is essential for the movement of the gastrointestinal tract. Interstitial cells of Cajal (ICC) lie adjacent to smooth muscle layers and are implicated as the pacemaker cells. However, the pace making mechanism remains unclear. To study the intercellular interaction during electrical rhythm generation, we visualized changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle cells and myenteric ICC within segments of mouse ileum loaded with a fluorescent Ca²⁺ indicator, fluo-3. We observed rhythmic [Ca²⁺]_i changes in longitudinal smooth muscle cells travelling rapidly through the smooth muscle cell layer. Between the rhythmic Ca²⁺ transients, we found brief Ca²⁺ transients localized to small areas within smooth muscle cells. The amplitude but not the periodicity of rhythmic [Ca²⁺]_i transients in both cell types was partially inhibited by nicardipine, an L-type Ca²⁺ channel antagonist, suggesting that the rhythmic [Ca²⁺]_i transients reflect membrane potential depolarizations corresponding to both slow waves and triggered Ca²⁺ spikes. Longitudinal smooth muscle cells and myenteric ICC showed synchronous spontaneous [Ca²⁺]_i transients in eight out of 21 ileac preparations analysed. In the remaining preparations, the synchrony between ICC and smooth muscle cells was absent, although the rhythmicity of the smooth muscle cells was not disturbed. These results suggest that myenteric ICC may play multiple roles including pace making for physiological bowel movement.     

Spatial Segregation and Interaction of Calcium Signalling Mechanisms in Rat Hippocampal CA1 Pyramidal Neurons

Postsynaptic [Ca²⁺]_i increases result from Ca²⁺ entry through ligand-gated channels, entry through voltage-gated channels, or release from intracellular stores. We found that these sources have distinct spatial distributions in hippocampal CA1 pyramidal neurons. Large amplitude regenerative release of Ca²⁺ from IP₃-sensitive stores in the form of Ca²⁺ waves were found almost exclusively on the thick apical shaft. Smaller release events did not extend more than 15 μm into the oblique dendrites. These synaptically activated regenerative waves initiated at points where the stimulated oblique dendrites branch from the apical shaft. In contrast, NMDA receptor-mediated increases were observed predominantly in oblique dendrites where spines are found at high density. These [Ca²⁺]_i increases were typically more than eight times larger than [Ca²⁺]_i from this source on the main aspiny apical shaft. Ca²⁺ entry through voltage-gated channels, activated by backpropagating action potentials, was detected at all dendritic locations. These mechanisms were not independent. Ca²⁺ entry through NMDA receptor channels or voltage-gated channels (as previously demonstrated) synergistically enhanced Ca²⁺ release generated by mGluR mobilization of IP₃.     

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 m m , bound to fatty acid-free albumin) to intact islets exposed to 15 m m glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 m m ) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 m m palmitate, an effect that was abolished in the presence of 10 μ m isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.     

The Effect of Calcium Mobilization on LPS-Induced IL-1β Production Depends on the Differentiation Stage of the Monocytes/Macrophages

The role of elevated intracellular Calcium concentration [Ca²⁺]_i in the LPS-induced activation of interleukin-1β (IL-1β) production was examined in cells representing different stages of myeloid differentiation (undifferentiated monocytic leukaemia cell line THP-1, THP-1 cells induced to adherent, macrophage-like cells by phorbol ester treatment and normal peripheral blood-derived adherent monocytes). LPS did not elevate the [Ca²⁺]¹, as measured by the Fura-2 fluorescence technique. When these cells were stimulated with LPS in the presence of the calcium ionophore A23187, a clear increase in the IL- 1β protein production was observed in the undifferentiated THP-1 cells but not in the more differentiated cell types. This ionophore-induced increase was also seen in the IL-1β mRNA levels. Thus these data confirm the previous findings demonstrating that elevation of (Ca-²⁺]₁ is not involved in the LPS-dependent signal transmission. However, the LPS-induced signals are greatly potentiated by the elevated [Ca²⁺]_i, but only in undifferentiated monocytic cells.     

Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl− transport in biliary epithelial cells through a PKCζ-dependent pathway

ATP in bile is a potent secretogogue, stimulating cholangiocyte Cl⁻ and fluid secretion via binding to membrane P2 receptors, though the physiological stimuli involved in biliary ATP release are unknown. The goal of the present studies was to determine the potential role of fluid flow in biliary ATP release and secretion. In both human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers, exposure to flow increased relative ATP release which was proportional to the shear stress. In parallel studies, shear was associated with an increase in [Ca²⁺]_i and membrane Cl⁻ permeability, which were both dependent on extracellular ATP and P2 receptor stimulation. Flow-stimulated ATP release was dependent on [Ca²⁺]_i, exhibited desensitization with repetitive stimulation, and was regulated by PKCζ. In conclusion, both human and rat biliary cells exhibit flow-stimulated, PKCζ-dependent, ATP release, increases in [Ca²⁺]_i and Cl⁻ secretion. The finding that fluid flow can regulate membrane transport suggests that mechanosensitive ATP release may be a key regulator of biliary secretion and an important target to modulate bile flow in the treatment of cholestatic liver diseases.     

Sensitivity limits for voltage control of P2Y receptor-evoked Ca2+ mobilization in the rat megakaryocyte

G-protein-coupled receptor signalling has been suggested to be voltage dependent in a number of cell types; however, the limits of sensitivity of this potentially important phenomenon are unknown. Using the non-excitable rat megakaryocyte as a model system, we now show that P2Y receptor-evoked Ca²⁺ mobilization is controlled by membrane voltage in a graded and bipolar manner without evidence for a discrete threshold potential. Throughout the range of potentials studied, the peak increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in response to depolarization was always larger than the maximal reduction in [Ca²⁺]_i following an equivalent amplitude hyperpolarization. Significant [Ca²⁺]_i increases were observed in response to small amplitude (<5 mV, 5 s duration) or short duration (25 ms, 135 mV) depolarizations. Individual cardiac action potential waveforms were also able to repeatedly potentiate P2Y receptor-evoked Ca²⁺ release and the response to trains of normally paced stimuli fused to generate prolonged [Ca²⁺]_i increases. Furthermore, elevation of the temperature to physiological levels (36°C) resulted in a more sustained depolarization-evoked Ca²⁺ increase compared with more transient or oscillatory responses at 20–24°C. The ability of signalling via a G-protein-coupled receptor to be potentiated by action potential waveforms and small amplitude depolarizations has broad implications in excitable and non-excitable tissues.     

Interplay between SERCA and sarcolemmal Ca2+ efflux pathways controls spontaneous release of Ca2+ from the sarcoplasmic reticulum in rat ventricular myocytes

Waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. We have investigated the effects of inhibiting the SR Ca²⁺-ATPase (SERCA) with the reversible inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on the properties of these waves. Cardiac myocytes were voltage clamped at a constant potential between −65 and −40 mV and spontaneous waves evoked by increasing external Ca²⁺ concentration to 4 m m . Application of 100 μ m TBQ decreased the frequency of waves. This was associated with increases of resting [Ca²⁺]_i, the time constant of decay of [Ca²⁺]_i and the integral of the accompanying Na⁺–Ca²⁺ exchange current. There was also a decrease in propagation velocity of the waves. There was an increase of the calculated Ca²⁺ efflux per wave. The SR Ca²⁺ content when a wave was about to propagate decreased to 91.7 ± 3.2%. The period between waves increased in direct proportion to the Ca²⁺ efflux per wave meaning that TBQ had no effect on the Ca²⁺ efflux per unit time. We conclude that (i) decreased wave frequency is not a direct consequence of decreased Ca²⁺ pumping by SERCA between waves but, rather, to more Ca²⁺ loss on each wave; (ii) inhibiting SERCA increases the chance of spontaneous Ca²⁺ release propagating at a given SR content.     

Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current ( I _ti). I _ti is the result of three different [Ca]_i-sensitive currents: the Na⁺–Ca²⁺ exchange current, a Ca²⁺-activated chloride current and a Ca²⁺-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca²⁺-activated non-selective cation channel (NSC_Ca) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na⁺ and K⁺) and was slightly permeable to Ca²⁺ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K _d for [Ca²⁺]_i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC_Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca²⁺] overload and thus may be implicated in the genesis of arrhythmias.