Mitochondrial Ca(2+) and apoptosis

Mitochondria are key decoding stations of the apoptotic process. In support of this view, a large body of experimental evidence has unambiguously revealed that, in addition to the well-established function of producing most of the cellular ATP, mitochondria play a fundamental role in triggering apoptotic cell death. Various apoptotic stimuli cause the release of specific mitochondrial pro-apoptotic factors into the cytosol. The molecular mechanism of this release is still controversial, but there is no doubt that mitochondrial calcium (Ca(2+)) overload is one of the pro-apoptotic ways to induce the swelling of mitochondria, with perturbation or rupture of the outer membrane, and in turn the release of mitochondrial apoptotic factors into the cytosol. Here, we review as different proteins that participate in mitochondrial Ca(2+) homeostasis and in turn modulate the effectiveness of Ca(2+)-dependent apoptotic stimuli. Strikingly, the final outcome at the cellular level is similar, albeit through completely different molecular mechanisms: a reduced mitochondrial Ca(2+) overload upon pro-apoptotic stimuli that dramatically blunts the apoptotic response.     

Intracellular Ca(2+) pools in neuronal signalling

Different intracellular pools participate in generating Ca(2+) signals in neuronal cells and in shaping their spatio-temporal patterns. They include the endoplasmic reticulum (endowed with different classes of Ca(2+) channels, with distinct functional properties and highly defined expression patterns in the brain), the Golgi apparatus, and the mitochondria. The release of Ca(2+) from intracellular pools plays an important role in controlling processes such as neurite outgrowth, synaptic plasticity, secretion and neurodegeneration.     

Intracellular Ca(2+) release channels in evolution

Intracellular Ca(2+)-release channels (ICRCs) form a superfamily of genes that encompasses two distinct subfamilies: the inositol trisphosphate receptor and the ryanodine receptor genes, which encode the largest ion channels known today. During evolution from nematodes to man, mechanisms of gene duplication and divergence have increased the number of known ICRC genes, which have been gradually co-opted to contribute to the increasing complexity of intracellular Ca(2+) signalling required for regulation of specialised eukaryotic cell activities.     

Tyrosine phosphorylation is involved in Ca(2+)entry in human gingival fibroblasts

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.     

A new type of Ca(2+) channel blocker that targets Ca(2+) sensors and prevents Ca(2+)-mediated apoptosis.

Calmodulin (CaM), as well as other Ca(2+) binding motifs (i.e., EF hands), have been demonstrated to be Ca(2+) sensors for several ion channel types, usually resulting in an inactivation in a negative feedback manner. This provides a novel target for the regulation of such channels. We have designed peptides that interact with EF hands of CaM in a specific and productive manner. Here we have examined whether these peptides block certain Ca(2+)-permeant channels and inhibit biological activity that is dependent on the influx of Ca(2+). We found that these peptides are able to enter the cell and directly, as well as indirectly (through CaM), block the activity of glutamate receptor channels in cultured neocortical neurons and a nonselective cation channel in Jurkat T cells that is activated by HIV-1 gp120. As a consequence, apoptosis mediated by an influx of Ca(2+) through these channels was also dose-dependently inhibited by these novel peptides. Thus, this new type of Ca(2+) channel blocker may have utility in controlling apoptosis due to HIV infection or neuronal loss due to ischemia.     

Intracellular Ca(2+) channel immunoreactivity in neuroendocrine axon terminals

The concentration of neuroendocrine terminals in the neurohypophysis facilitates the identification and localization of Ca(2+) channel subtypes near neuroendocrine release sites. Immunoblots of rat neurohypophysial tissue identified the alpha(1)1.3, alpha(1)2.1, alpha(1)2.2, and alpha(1)2.3 Ca(2+) channel subunits. Immunofluorescence staining of axon terminal plasma membranes was weak, suggesting that Ca(2+) channels are dispersed. This contrasts with the highly punctate alpha(1)2.2 immunoreactivity in bovine chromaffin cells; the neurohypophysial terminals may therefore lack the specialized release zones found in those cells. Immunofluorescence and immunogold labeling identify dense core granule-like structures in the terminal cytoplasm containing multiple Ca(2+) channel types. Ca(2+) channels in internal membranes may play an important role in channel targeting and distribution in neuroendocrine cells.     

Intracellular Ca(2+) release as irreversible Markov process

In striated muscles, intracellular Ca(2+) release is tightly controlled by the membrane voltage sensor. Ca(2+) ions are necessary mediators of this control in cardiac but not in skeletal muscle, where their role is ill-understood. An intrinsic gating oscillation of Ca(2+) release-not involving the voltage sensor-is demonstrated in frog skeletal muscle fibers under voltage clamp. A Markov model of the Ca(2+) release units is shown to reproduce the oscillations, and it is demonstrated that for Markov processes to have oscillatory transients, its transition rates must violate thermodynamic reversibility. Such irreversibility results in permanent cycling of the units through a ring of states, which requires a source of free energy. Inhibition of the oscillation by 20 to 40 mM EGTA or partial depletion of Ca(2+) in the sarcoplasmic reticulum (SR) identifies the SR [Ca(2+)] gradient as the energy source, and indicates a location of the critical Ca(2+)-sensing site at distances greater than 35 nm from the open channel. These results, which are consistent with a recent demonstration of irreversibility in gating of cardiac Ca(2+) sparks, (Wang, S.-Q., L.-S. Song, L. Xu, G. Meissner, E. G. Lakatta, E. Ríos, M. D. Stern, and H. Cheng. 2002. Biophys. J. 83:242-251) exemplify a cell-wide oscillation caused by coupling between ion permeation and channel gating.     

Nucleoplasmic Ca(2+)loading is regulated by mobilization of perinuclear Ca(2+)

Regulation of nucleoplasmic calcium (Ca(2+)) concentration may occur by the mobilization of perinuclear luminal Ca(2+)pools involving specific Ca(2+)pumps and channels of both inner and outer perinuclear membranes. To determine the role of perinuclear luminal Ca(2+), we examined freshly cultured 10 day-old embryonic chick ventricular cardiomyocytes. We obtained evidence suggesting the existence of the molecular machinery required for the bi-directional Ca(2+)fluxes using confocal imaging techniques. Embryonic cardiomyocytes were probed with antibodies specific for ryanodine-sensitive Ca(2+)channels (RyR2), sarco/endoplasmic reticulum Ca(2+)ATPase (SERCA2)-pumps, and fluorescent BODIPY derivatives of ryanodine and thapsigargin. Using immunocytochemistry techniques, confocal imaging showed the presence of RyR2 Ca(2+)channels and SERCA2-pumps highly localized to regions surrounding the nucleus, referable to the nuclear envelope. Results obtained from Fluo-3, AM loaded ionomycin-perforated embryonic cardiomyocytes demonstrated that gradual increases of extranuclear Ca(2+)from 100 to 1600 nM Ca(2+)was localized to the nucleus. SERCA2-pump inhibitors thapsigargin and cyclopiazonic acid showed a concentration-dependent inhibition of nuclear Ca(2+)loading. Furthermore, ryanodine demonstrated a biphasic concentration-dependence upon active nuclear Ca(2+)loading. The concomitant addition of thapsigargin or cyclopiazonic acid with ryanodine at inhibitory concentrations caused an significant increase in nuclear Ca(2+)loading at low concentrations of extranuclear added Ca(2+). Our results show that the perinuclear lumen in embryonic chick ventricular cardiomyocytes is capable of autonomously regulating nucleoplasmic Ca(2+)fluxes.     

Cytosolic Ca(2+) signal is involved in regulating UV-induced apoptosis in hela cells

Results of recent studies using BAPTA/AM have raised a serious question on whether Ca(2+) signal is truly involved in regulating the progression of apoptosis. To resolve this question, we examined the differential effects of three different Ca(2+) signaling blockers (BAPTA/AM, membrane-impermeant BAPTA, and heparin) on UV-induced apoptosis in HeLa cells. We found that although the membrane-permeable form of BAPTA (i.e., BAPTA/AM) could not inhibit cell death, the membrane-impermeant form of BAPTA, loaded into the cytosol by electroporation, clearly protected cells from entering apoptosis. Furthermore, when we injected heparin to block Ca(2+) release from the endoplasmic reticulum (ER) to cytosol, apoptosis was greatly suppressed. These findings strongly suggest that elevation of cytosolic Ca(2+) is part of the signal that drives the progression of apoptosis. The negative result of BAPTA/AM is probably due to its dual effect on subcellular Ca(2+) distribution; besides suppressing the Ca(2+) elevation in cytosol, BAPTA/AM can also enter into the ER to reduce the free Ca(2+) level there. The depletion of Ca(2+) in ER is believed to stimulate apoptosis and thus would counterbalance the protection effect of BAPTA/AM in suppressing the cytosolic Ca(2+) elevation.     

Intracellular Ca(2+) dynamics and sarcomere length in single ventricular myocytes

The measurements of the sarcomere length in dissociated cardiac ventricular myocytes are discussed using mainly our own experimental data. The striation periodicity of these unloaded cells was found to be that which is to be expected of a myocyte free of the ultrastructural constraints imposed upon it by the normal syncytial matrix of the ventricular wall. The sarcomere length and [Ca(2+)] relationship was consistent as expected from the intact tissue, when it was measured soon after partial rupturing the cell membrane. Miniature fluctuations of individual sarcomere length were demonstrated during rest, which was augmented by the Ca(2+) overload. The [Ca(2+)] could be estimated from the measurements of sarcomere length during the positive staircase of contraction. The usefulness of the optical measurement of sarcomere pattern was indicated.     

Intracellular Ca(2+) dynamics and the stability of ventricular tachycardia

Ventricular fibrillation (VF), the major cause of sudden cardiac death, is typically preceded by ventricular tachycardia (VT), but the mechanisms underlying the transition from VT to VF are poorly understood. Intracellular Ca(2+) overload occurs during rapid heart rates typical of VT and is also known to promote arrhythmias. We therefore studied the role of intracellular Ca(2+) dynamics in the transition from VT to VF, using a combined experimental and mathematical modeling approach. Our results show that 1) rapid pacing of rabbit ventricular myocytes at 35 degrees C led to increased intracellular Ca(2+) levels and complex patterns of action potential (AP) configuration and the intracellular Ca(2+) transients; 2) the complex patterns of the Ca(2+) transient arose directly from the dynamics of intracellular Ca(2+) cycling, and were not merely passive responses to beat-to-beat alterations in AP; 3) the complex Ca(2+) dynamics were simulated in a modified version of the Luo-Rudy (LR) ventricular action potential with improved intracellular Ca(2+) dynamics, and showed good agreement with the experimental findings in isolated myocytes; and 4) when incorporated into simulated two-dimensional cardiac tissue, this action potential model produced a form of spiral wave breakup from VT to a VF-like state in which intracellular Ca(2+) dynamics played a key role through its influence on Ca(2+)-sensitive membrane currents such as I(Ca), I(NaCa), and I(ns(Ca)). To the extent that spiral wave breakup is useful as a model for the transition from VT to VF, these findings suggest that intracellular Ca(2+) dynamics may play an important role in the destabilization of VT and its degeneration into VF.     

Intracellular Ca(2+) oscillations induced by over-expressed Ca(V)3.1 T-type Ca(2+) channels in NG108-15 cells

T-type Ca²⁺ channel family includes three subunits Ca_V3.1, Ca_V3.2 and Ca_V3.3 and have been shown to control burst firing and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Here, we investigated whether Ca_V3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca_V3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca_V3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca²⁺]_i oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around −61 mV. [Ca²⁺]_i oscillations were critically dependent on Ca²⁺ influx through Ca_V3.1 channels and did not involve Ca²⁺ release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca_V3.1 channels. The trigger of the oscillations was the Ca_V3.1 window current. This current induced continuous [Ca²⁺]_i increase at −60 mV that depolarized the cells and triggered MPOs. Shifting the Ca_V3.1 window current potential range by increasing the external Ca²⁺ concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I_h) was not required to induce MPOs, but when expressed together with Ca_V3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca_V3.1 window current is critical in triggering intrinsic electrical and [Ca²⁺]_i oscillations.     

Intracellular Ca(2+) and Zn(2+) signals during monochloramine-induced oxidative stress in isolated rat colon crypts

During acute exacerbations of inflammatory bowel diseases, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the colon mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH(2)Cl), to serve as agonists of Ca(2+) and Zn(2+) accumulation within the colonocyte. Individual colon crypts prepared from Sprague-Dawley rats were mounted in perfusion chambers after loading with fluorescent reporters fura 2-AM and fluozin 3-AM. These reporters were characterized, in situ, for responsiveness to Ca(2+) and Zn(2+) in the cytoplasm. Responses to different concentrations of NH(2)Cl (50, 100, and 200 microM) were monitored. Subsequent studies were designed to identify the sources and mechanisms of NH(2)Cl-induced increases in Ca(2+) and Zn(2+) in the cytoplasm. Exposure to NH(2)Cl led to dose-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in the range of 200-400 nM above baseline levels. Further studies indicated that NH(2)Cl-induced accumulation of Ca(2+) in the cytoplasm is the result of release from intracellular stores and basolateral entry of extracellular Ca(2+) through store-operated channels. In addition, exposure to NH(2)Cl resulted in dose-dependent and sustained increases in intracellular Zn(2+) concentration ([Zn(2+)](i)) in the nanomolar range. These alterations were neutralized by dithiothreitol, which shields intracellular thiol groups from oxidation. We conclude that Ca(2+)- and Zn(2+)-handling proteins are susceptible to oxidation by chloramines, leading to sustained, but not necessarily toxic, increases in [Ca(2+)](i) and [Zn(2+)](i). Under certain conditions, NH(2)Cl may act not as a toxin but as an agent that activates intracellular signaling pathways.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis

A variety of stimuli utilize an increase of cytosolic free Ca²⁺ concentration as a second messenger to transmit signals, through Ca²⁺ release from the endoplasmic reticulum or opening of plasma membrane Ca²⁺ channels. Mitochondria contribute to the tight spatiotemporal control of this process by accumulating Ca²⁺, thus shaping the return of cytosolic Ca²⁺ to resting levels. The rise of mitochondrial matrix free Ca²⁺ concentration stimulates oxidative metabolism; yet, in the presence of a variety of sensitizing factors of pathophysiological relevance, the matrix Ca²⁺ increase can also lead to opening of the permeability transition pore (PTP), a high conductance inner membrane channel. While transient openings may serve the purpose of providing a fast Ca²⁺ release mechanism, persistent PTP opening is followed by deregulated release of matrix Ca²⁺, termination of oxidative phosphorylation, matrix swelling with inner membrane unfolding and eventually outer membrane rupture with release of apoptogenic proteins and cell death. Thus, a rise in mitochondrial Ca²⁺ can convey both apoptotic and necrotic death signals by inducing opening of the PTP. Understanding the signalling networks that govern changes in mitochondrial free Ca²⁺ concentration, their interplay with Ca²⁺ signalling in other subcellular compartments, and regulation of PTP has important implications in the fine comprehension of the main biological routines of the cell and in disease pathogenesis.     

Neuronal galvanotropism is independent of external Ca(2+) entry or internal Ca(2+) gradients

The mechanism by which growing neurites sense and respond to small applied electrical fields is not known, but there is some evidence that the entry of Ca(2+) from the external medium, with the subsequent formation of intracellular Ca(2+) gradients, is important in this process. We have employed two approaches to test this idea. Xenopus spinal neurites were exposed to electrical fields in a culture medium in which Ca(2+) was chelated to very low levels compared to the normal extracellular concentration of 2 mM. In other experiments, loading the neurites with the calcium buffer, 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), disrupted the putative internal Ca(2+) gradients, and the effects on the electrical response were determined. Fields of 100 mV/mm were applied for 12 h, and no difference was detected in the cathodal turning response between the treated neurites and the untreated controls. Using the Differential Growth Index (DGI), an asymmetry index, to quantitate the turning response, we recorded DGIs of -0.64, -0.65, and -0.62 for control cells, cells in Ca(2+)-free medium, and cells preloaded with BAPTA, respectively. Furthermore, we detected an increase in neurite length for those neurons cultured in Ca(2+)-free medium; they were 1.5-1.7 times as long as neurites from neurons cultured in normal Ca(2+) medium. Likewise, we found that BAPTA-loaded neurites were longer than control neurites. Our data indicate that neuronal galvanotropism is independent of the entry of external Ca(2+) or of internal Ca(2+) gradients. Both cell-permeant agonistic and antagonistic analogs of cyclic 3',5'-adenosine monophosphate (cAMP) increased the response to applied electrical fields.     

Hormone-stimulated Mg(2+) accumulation into rat hepatocytes: a pathway for rapid Mg(2+) and Ca(2+) redistribution

Many diseases such as cardiac arrhythmia, diabetes, and chronic alcoholism are associated with a marked decrease of plasma and parenchymal Mg(2+), and Mg(2+) administration is routinely used therapeutically. This study uses isolated rat hepatocytes to ascertain if and under which conditions increases in extracellular Mg(2+) result in an increase in intracellular Mg(2+). In the absence of stimulation, changing extracellular Mg(2+) had no effect on total cellular Mg(2+) content. By contrast, carbachol or vasopressin administration promoted an accumulation of Mg(2+) that increased cellular Mg(2+) content by 13.2 and 11.8%, respectively, and stimulated Mg(2+) uptake was unaffected by the absence of extracellular Ca(2+). Mg(2+) efflux resulting from stimulation of alpha- or beta-adrenergic receptors operated with a Mg(2+):Ca(2+) exchange ratio of 1. These data indicate that cellular Mg(2+) uptake can occur rapidly and in large amounts, through a process distinct from Mg(2+) release, but operating only upon specific hormonal stimulation.     

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.     

Intracellular Ca(2+) regulates responsiveness of cardiac L-type Ca(2+) current to protein kinase A: role of calmodulin

The goal of this study was to determine whether the protein kinase A (PKA) responsiveness of the cardiac L-type Ca(2+) current (ICa) is affected during transient increases in intracellular Ca(2+) concentration. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on aligned collagen thin gels. When measured in 1 or 2 mM Ca(2+) external solution, the aligned myocytes displayed a large ICa that was weakly regulated (20% increase) during stimulation of PKA by 2 microM forskolin. In contrast, application of forskolin caused a 100% increase in ICa when the external Ca(2+) concentration was reduced to 0.5 mM or replaced with Ba(2+). This Ca(2+)-dependent inhibition was also observed when the cells were treated with 1 microM isoproterenol, 100 microM 3-isobutyl-1-methylxanthine, or 500 microM 8-bromo-cAMP. The responsiveness of ICa to PKA was restored during intracellular dialysis with a calmodulin (CaM) inhibitory peptide but not during treatment with inhibitors of protein kinase C, Ca(2+)/CaM-dependent protein kinase, or calcineurin. Adenoviral-mediated expression of a CaM molecule with mutations in all four Ca(2+)-binding sites also increased the PKA sensitivity of ICa. Finally, adult mouse ventricular myocytes displayed a greater response to forskolin and cAMP in external Ba(2+). Thus Ca(2+) entering the myocyte through the voltage-gated Ca(2+) channel regulates the PKA responsiveness of ICa.     

Intracellular Mg(2+) enhances the function of BK-type Ca(2+)-activated K(+) channels.

BK channels modulate neurotransmitter release due to their activation by voltage and Ca(2+). Intracellular Mg(2+) also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg(2+) blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg(2+) activates mslo1 BK channels independently of Ca(2+) and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca(2+) binding sites in the tail, is not activated by Mg(2+). However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg(2+) sensitivity similar to mslo1 channels, indicating that Mg(2+) sites differ from Ca(2+) sites. We discovered that Mg(2+) also binds to Ca(2+) sites and competitively inhibits Ca(2+)-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg(2+) under physiological conditions is to enhance BK channel function.