Leak Ca2 Research Articles

Effects of Sarcolemmal Background Ca2+ Entry and Sarcoplasmic Ca2+ Leak Currents on Electrophysiology and Ca2+ Transients in Human Ventricular Cardiomyocytes: A Computational Comparison.

The intricate regulation of the compartmental Ca2+ concentrations in cardiomyocytes is critical for electrophysiology, excitation-contraction coupling, and other signaling pathways. Research into the complex signaling pathways is motivated by cardiac pathologies including arrhythmia and maladaptive myocyte remodeling, which result from Ca2+ dysregulation. Of interest to this investigation are two types of Ca2+ currents in cardiomyocytes: 1) background Ca2+ entry, i.e., Ca2+ transport across the sarcolemma from the extracellular space into the cytosol, and 2) Ca2+ leak from the sarcoplasmic reticulum (SR) across the SR membrane into the cytosol. Candidates for the ion channels underlying background Ca2+ entry and SR Ca2+ leak channels include members of the mechano-modulated transient receptor potential (TRP) family. We used a mathematical model of a human ventricular myocyte to analyze the individual contributions of background Ca2+ entry and SR Ca2+ leak to the modulation of Ca2+ transients and SR Ca2+ load at rest and during action potentials. Background Ca2+ entry exhibited a positive relationship with both [Ca2+]i and [Ca2+]SR. Modulating SR Ca2+ leak had opposite effects of background Ca2+ entry. Effects of SR Ca2+ leak on Ca2+ were particularly pronounced at lower pacing frequency. In contrast to the pronounced effects of background and leak Ca2+ currents on Ca2+ concentrations, the effects on cellular electrophysiology were marginal. Our studies provide quantitative insights into the differential modulation of compartmental Ca2+ concentrations by the background and leak Ca2+ currents. Furthermore, our studies support the hypothesis that TRP channels play a role in strain-modulation of cardiac contractility. In summary, our investigations shed light on the physiological effects of the background and leak Ca2+ currents and their contribution to the development of disease caused by Ca2+ dysregulation.

Mg2+ Is a Missing Link in Plant Cell Ca2+ Signalling and Homeostasis-A Study on Vicia faba Guard Cells.

Hyperpolarization-activated calcium channels (HACCs) are found in the plasma membrane and tonoplast of many plant cell types, where they have an important role in Ca2+-dependent signalling. The unusual gating properties of HACCs in plants, i.e., activation by membrane hyperpolarization rather than depolarization, dictates that HACCs are normally open in the physiological hyperpolarized resting membrane potential state (the so-called pump or P-state); thus, if not regulated, they would continuously leak Ca2+ into cells. HACCs are permeable to Ca2+, Ba2+, and Mg2+; activated by H2O2 and the plant hormone abscisic acid (ABA); and their activity in guard cells is greatly reduced by increasing amounts of free cytosolic Ca2+ ([Ca2+]Cyt), and hence closes during [Ca2+]Cyt surges. Here, we demonstrate that the presence of the commonly used Mg-ATP inside the guard cell greatly reduces HACC activity, especially at voltages ≤ −200 mV, and that Mg2+ causes this block. Therefore, we firstly conclude that physiological cytosolic Mg2+ levels affect HACC gating and that channel opening requires either high negative voltages (≥−200 mV) or displacement of Mg2+ away from the immediate vicinity of the channel. Secondly, based on structural comparisons with a Mg2+-sensitive animal inward-rectifying K+ channel, we propose that the likely candidate HACCs described here are cyclic nucleotide gated channels (CNGCs), many of which also contain a conserved diacidic Mg2+ binding motif within their pores. This conclusion is consistent with the electrophysiological data. Finally, we propose that Mg2+, much like in animal cells, is an important component in Ca2+ signalling and homeostasis in plants.

The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: possible physiological significance of specific cleavage by caspase 3.

The type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is an intracellular Ca(2+) channel protein that plays crucial roles in generating complex Ca(2+) signalling patterns. IP(3)R1 consists of three domains: a ligand-binding domain, a regulatory domain and a channel domain. In order to investigate the function of these domains in its gating machinery and the physiological significance of specific cleavage by caspase 3 that is observed in cells undergoing apoptosis, we utilized various IP(3)R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP(3)R1 or IP(3)R1 lacking the ligand-binding domain in HeLa and COS-7 cells had little effect on cells' responsiveness to an IP(3)-generating agonist ATP and Ca(2+) leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP-IP(3)R1-casp) or the channel domain alone (GFP-IP(3)R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca(2+) concentration. Interestingly, store-operated (-like) Ca(2+) entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca(2+) stores of cells expressing GFP-IP(3)R1-casp or GFP-IP(3)R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca(2+). We therefore propose that the channel domain of IP(3)R1 tends to remain open and that the large regulatory domain of IP(3)R1 is necessary to keep the channel domain closed. Thus cleavage of IP(3)R1 by caspase 3 may contribute to the increased cytosolic Ca(2+) concentration often observed in cells undergoing apoptosis. Finally, GFP-IP(3)R1-casp or GFP-IP(3)R1-ES can be used as a novel tool to deplete intracellular Ca(2+) stores.

Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2.

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.

Divergent effects of extracellular and intracellular alkalosis on Ca2+ entry pathways in vascular endothelial cells.

Modulation by alkalosis of basal leak Ca2+ entry and store-depletion-induced Ca2+ entry was investigated in the vascular endothelial cell line ECV 304. Ca2+ entry was monitored as the increase in the intracellular free Ca2+ concentration ([Ca2+]i) induced by elevation of the extracellular Ca2+ concentration. When ECV 304 cells were challenged with 100 nM thapsigargin in nominally Ca2+-free solution, [Ca2+]i increased transiently, and the increase in [Ca2+]i during a subsequent cumulative elevation of extracellular Ca2+ (from nominally Ca2+-free up to 5 mM) was markedly enhanced compared with non-stimulated cells (i.e. basal Ca2+ leak). Prolonged elevation of the extracellular pH (pHo) from 7.4 to 7.9 did not affect resting [Ca2+]i or the thapsigargin-induced [Ca2+]i transient evoked in nominally Ca2+-free solution, but increased leak Ca2+ entry as well as store-depletion-activated Ca2+ entry significantly. Basal Ca2+ leak and store-depletion-activated Ca2+ entry were enhanced either by acute elevation of pHo from 7.4 to 7.9 or by chronic alkalosis (pHo=7.9). Stimulation of Ca2+ entry by extracellular alkalosis was observed both in normal and in high extracellular K+ (110 mM) solution, suggesting that the effects of alkalosis are independent of membrane potential. The intracellular pH (pHi) increased slightly during both acute and chronic extracellular alkalosis (from 7.22+/-0.01 to 7.37+/-0.04 and 7. 45+/-0.05 respectively). Elevation of pHi to 7.60+/-0.06 at constant pHo by administration of 20 mM NH4Cl failed to stimulate, and in fact inhibited, store-depletion-activated Ca2+ entry. Our results demonstrate that a decrease in the extracellular but not the intracellular proton concentration promotes both basal and stimulated Ca2+ entry into endothelial cells.

Evidence for a Direct Inhibitory Effect of Extracellular H+on Store Depletion-Activated Ca2+Entry in Vascular Endothelial Cells

Modulation of store depletion-activated Ca2+entry by acidosis was investigated in ECV304 endothelial cells. Lowering extracellular pH from 7.4 to 6.9 markedly suppressed Ca2+entry elicited by direct depletion of Ca2+stores with thapsigargin (100 nM), but did not significantly affect leak Ca2+entry. Acidosis diminished thapsigargin-induced Ca2+entry by 53.7 ± 7.8% at 2.5 mM extracellular Ca2+. A similar degree of inhibition was observed in cells depolarized by high extracellular K+(100 mM). Reduction of extracellular pH from 7.4 to 6.9 was associated with a decrease in intracellular pH from 7.23 ± 0.01 to 7.01 ± 0.03. Propionate (20 mM) caused a reduction of intracellular pH to 6.97 ± 0.02, but failed to suppress store depletion-activated Ca2+entry at 2.5 mM extracellular Ca2+significantly. Our results suggest that an increase in extracellular proton concentration inhibits store depletion-activated Ca2+entry through a direct, membrane potential-independent effect on the plasmalemmal Ca2+channel.

Non‐selective cation current of guinea‐pig endocardial endothelial cells.

1. Endocardial endothelial (EE) cells, isolated by the enzymatic treatment of guinea-pig heart, were used for whole-cell voltage clamp experiments. 2. The inward rectifier K+ current was observed in about half of the experiments. The contribution of Ca(2+)-dependent K+ current to the resting membrane conductance was also suggested. 3. After the K+ conductances were suppressed, removal of external Na+ revealed an inward cation current (1.2 pA pF-1, at -45 mV), whose slope conductance was a saturable function of external Na+ concentration. When Na+ was totally replaced by various monovalent cations, the order of the membrane conductances was K+ > Rb+ > Cs+ > Na+ > Li+. 4. This basal non-selective cation current was blocked by either Gd3+ or La3+, and showed slight outward rectification. 5. Addition of 20 mM Ca2+ or Ba2+, but not Mg2+ or Mn2+, to the Na(+)-free solution, induced an inward current, indicating that this current possesses a significant Ca2+ permeability. 6. In approximately 15% of the experiments, ATP and histamine induced another type of non-selective cation current, which showed different ion selectivity (Na+ > K+, Cs+) and rectification (inward). 7. The basal non-selective cation current is responsible for both the low resting potential and the leak Ca2+ influx of EE cells.

Erythrocyte membrane abnormalities in sickle cell disease.

A large fraction of circulating sickle red cells contain one or more large vesicles which are not found in normal erythrocytes. These vesicles contain very high levels of Ca2+, and probably account for the long-known elevation of cellular Ca2+ in sickle cells. These vesicles contain the plasma membrane CaATPase and leak Ca2+ by a nitrendipine-sensitive pathway. The question of whether an abnormal endocytic process occurs in sickle cells which could give rise to these vesicles was examined using the nonspecific endocytic marker Lucifer yellow (LY). The kinetics of formation of LY-labeled endocytic vesicles in sickle cells includes a slow component which is not found in normal (or sickle) reticulocytes. In addition, the number of sickle cells in which endocytosis can be demonstrated with this nonspecific marker consistently exceeds the number which can be labeled with markers of the receptor-mediated endocytic process. The results suggest that a slow, abnormal endocytosis takes place in sickle cells which may be the source of the Ca2(+)-containing vesicles, and therefore of the elevated Ca2+ levels characteristic of the circulating cells in this disease.