Mitogen-induced changes in Ca2+ permeability are not mediated by voltage-gated K+ channels

Binding of mitogenic lectins to T lymphocytes results in elevated cytoplasmic Ca2+ concentrations ([Ca2+]i). This change in [Ca2+]i is thought to be essential for cellular proliferation. In addition, the lectins increase the conductance to K+ through voltage-sensitive channels. Based on the inhibitory effect of K+ channel blockers on lectin-induced mitogenesis, it has been suggested that Ca2+ could enter the cells through these activated K+ channels (Chandy, K. G., De Coursey, T. E., Cahalan, M. D., McLaughlin, C., and Gupta, S. (1984) J. Exp. Med. 160, 369-385; Chandy, K. G., De Coursey, T. E., Cahalan, M. D., and Gupta, S. (1985) J. Clin. Immunol. 5, 1-5). This hypothesis was tested experimentally by measuring the effect of activation or blockade of K+ channels on [Ca2+]i using quin-2 and indo-1 and by determining the effect of K+ channel blockers on lectin-induced proliferation. We found that: depolarization of the membrane, which is expected to open the K+ channels, failed to increase [Ca2+]i, K+ channel blockers such as tetraethylammonium and 4-aminopyridine had only a marginal effect on the lectin-induced increase in [Ca2+]i, and the inhibitory effect of K+ channel blockers on proliferation was found to be nonspecific, occurring also when proliferation was triggered by phorbol esters under conditions where [Ca2+]i is not elevated. It is concluded that the lectin-induced changes in [Ca2+]i are not mediated by the opening of voltage-gated K+ channels.     

Rapid effects of 17beta-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells

The control of calcium homeostasis is essential for cell survival and is of crucial importance for several physiological functions. The discovery of the epithelial calcium channel Transient Receptor Potential Vaniloid (TRPV6) in intestine has uncovered important Ca(2+) absorptive pathways involved in the regulation of whole body Ca(2+) homeostasis. The role of steroid hormone 17beta-estradiol (E(2)), in [Ca(2+)](i) regulation involving TRPV6 has been only limited at the protein expression levels in over-expressing heterologous systems. In the present study, using a combination of calcium-imaging, whole-cell patch-clamp techniques and siRNA technology to specifically knockdown TRPV6 protein expression, we were able to (i) show that TRPV6 is natively, rather than exogenously, expressed at mRNA and protein levels in human T84 colonic cells, (ii) characterize functional TRPV6 channels and (iii) demonstrate, for the first time, the rapid effects of E(2) in [Ca(2+)](i) regulation involving directly TRPV6 channels in T84 cells. Treatment with E(2) rapidly (<5 min) enhanced [Ca(2+)](i) and this increase was partially but significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV6 protein expression. These results indicate that when cells are stimulated by E(2), Ca(2+) enters the cell through TRPV6 channels. TRPV6 channels in T84 cells contribute to the Ca(2+) entry/signalling pathway that is sensitive to 17beta-estradiol.     

Evidence for two distinct voltage-gated calcium channel currents in bovine adrenal glomerulosa cells

We analyzed inward Ca2+ currents in single bovine adrenal glomerulosa cell using whole-cell patch clamp techniques. Two types of voltage-gated Ca2+ channel currents were identified. One was a transient (T) type which decayed within 100 ms, characterized by a low threshold voltage (about -70 mv) similar to that seen in rat adrenal glomerulosa cells (Matsunaga, H. et al. (1987) Pflügers Arch. 408, 351-355.) Another was a long-lasting (L) type which shows a more positive threshold potential. The present results suggest that while T type Ca2+ channels may explain initial calcium influx in response to an elevation in extracellular K+, L type Ca2+ channels may allow sustained calcium influx which is necessary for sustained aldosterone secretion.     

Ion channels and membrane potential in stimulus-secretion coupling in adrenal paraneurons

Cultured bovine adrenal medulla cells have been shown to contain several different ion channels (Na+, Ca2+, acetylcholine receptor regulated) whose activation leads to the secretion of catecholamines. The pharmacology of these ion channels and their interactions during secretion have been examined. The mechanisms of agonist-induced calcium influx are of particular interest since this is an early obligatory event during secretion from the adrenal medulla. Data obtained on catecholamine release and 45Ca2+ uptake indicate that both voltage-dependent and voltage-independent calcium influx mechanisms operate in cultured bovine adrenal medulla cells. The significance of these results in understanding the mechanism of action of the physiological stimulus acetylcholine (Ach) will be discussed. The alkaloid channel neurotoxins D-600, batrachotoxin, veratridine, and aconitine were shown to exert a noncompetitive inhibitory effect on Ach-induced ion flux in adrenal medulla cells, presumably through an interaction with the nicotinic receptor regulated channel. Lipid-soluble neurotoxins may interact with multiple ion channels in nerve and muscle membrane.     

Remodelling of the endoplasmic reticulum during store-operated calcium entry

SOCE (store-operated calcium entry) is a ubiquitous cellular mechanism linking the calcium depletion of the ER (endoplasmic reticulum) to the activation of PM (plasma membrane) Ca2+-permeable channels. The activation of SOCE channels favours the entry of extracellular Ca2+ into the cytosol, thereby promoting the refilling of the depleted ER Ca2+ stores as well as the generation of long-lasting calcium signals. The molecules that govern SOCE activation comprise ER Ca2+ sensors [STIM1 (stromal interaction molecule 1) and STIM2], PM Ca2+-permeable channels {Orai and TRPC [TRP (transient receptor potential) canonical]} and regulatory Ca2+-sensitive cytosolic proteins {CRACR2 [CRAC (Ca2+ release-activated Ca2+ current) regulator 2]}. Upon Ca2+ depletion of the ER, STIM molecules move towards the PM to bind and activate Orai or TRPC channels, initiating calcium entry and store refilling. This molecular rearrangement is accompanied by the formation of specialized compartments derived from the ER, the pre-cER (cortical ER) and cER. The pre-cER appears on the electron microscope as thin ER tubules enriched in STIM1 that extend along microtubules and that are devoid of contacts with the PM. The cER is located in immediate proximity to the PM and comprises thinner sections enriched in STIM1 and devoid of chaperones that might be dedicated to calcium signalling. Here, we review the molecular interactions and the morphological changes in ER structure that occur during the SOCE process.     

The pore region of the skeletal muscle ryanodine receptor is a primary locus for excitation-contraction uncoupling in central core disease

Human central core disease (CCD) is caused by mutations/deletions in the gene that encodes the skeletal muscle ryanodine receptor (RyR1). Previous studies have shown that CCD mutations in the NH2-terminal region of RyR1 lead to the formation of leaky SR Ca2+ release channels when expressed in myotubes derived from RyR1-knockout (dyspedic) mice, whereas a COOH-terminal mutant (I4897T) results in channels that are not leaky to Ca2+ but lack depolarization-induced Ca2+ release (termed excitation-contraction [EC] uncoupling). We show here that store depletion resulting from NH2-terminal (Y523S) and COOH-terminal (Y4795C) leaky CCD mutant release channels is eliminated after incorporation of the I4897T mutation into the channel (Y523S/I4897T and Y4795C/I4897T). In spite of normal SR Ca2+ content, myotubes expressing the double mutants lacked voltage-gated Ca2+ release and thus exhibited an EC uncoupling phenotype similar to that of I4897T-expressing myotubes. We also show that dyspedic myotubes expressing each of seven recently identified CCD mutations located in exon 102 of the RyR1 gene (G4890R, R4892W, I4897T, G4898E, G4898R, A4905V, R4913G) behave as EC-uncoupled release channels. Interestingly, voltage-gated Ca2+ release was nearly abolished (reduced approximately 90%) while caffeine-induced Ca2+ release was only marginally reduced in R4892W-expressing myotubes, indicating that this mutation preferentially disrupts voltage-sensor activation of release. These data demonstrate that CCD mutations in exon 102 disrupt release channel permeation to Ca2+ during EC coupling and that this region represents a primary molecular locus for EC uncoupling in CCD.     

Disease-causing mutations of calcium channels

Many cellular functions are directly or indirectly regulated by the free cytosolic calcium concentration. Thus, calcium levels must be very tightly regulated in time and space. Intracellular calcium ions are essential second messengers and play a role in many functions including, action potential generation, neurotransmitter and hormone release, muscle contraction, neurite outgrowth, synaptogenesis, calcium-dependent gene expression, synaptic plasticity and cell death. Calcium ions that control cell activity can be supplied to the cell cytosol from two major sources: the extracellular space or intracellular stores. Voltage-gated and ligand-gated channels are the primary way in which Ca2+ ions enter from the extracellular space. The sarcoplasm reticulum (SR) in muscle and the endoplasmic reticulum in non-muscle cells are the main intracellular Ca2+ stores: the ryanodine receptor (RyR) and inositol-triphosphate receptor channels are the major contributors of calcium release from internal stores. Mutations of genes encoding calcium have been implicated in the etiology of a diverse group of nerve and muscle diseases. These mutations have been identified in humans, mice and other organisms. In this review, we will summarize calcium channelopathies of humans and mice. Of the ten calcium channel α1 subunits cloned and sequenced (see ref. 1), disease-causing mutations have been found in CaV1.4 and CaV2.1 in the nervous system, and CaV1.1 and CaV1.2 in muscle. Mutations in calcium channel auxiliary subunits (α2δ, β and γ) have also been associated with both human and/or mouse neurological diseases. The disease-causing mutations may provide new insight into the cell biological roles of calcium channels as well as into relationships between structure and function. In addition, understanding how the mutations affect the physiology of the cell could lead to advances in disease treatment by relieving symptoms or slowing the progression of the disease. However, due to the multifaceted functions of calcium in the cell, the correlation between molecular mutation, physiological alterations and disease etiology is neither straightforward nor easily understood. Since calcium is an important intracellular signaling molecule, altered calcium channel function can give rise to widespread changes in cellular function. Indeed, serious diseases result from mutations that cause trivial alterations of calcium currents analyzed in vitro.     

Mitochondria as biosensors of calcium microdomains

The notion that the agonist-dependent increases in intracellular Ca2+ concentration, on ubiquitous signalling mechanism, occur with a tightly regulated spatio-temporal pattern has become an established concept in modern cell biology. As a consequence, the concept is emerging that the recruitment of specific intracellular targets and effector system mechanisms depends on exposure to local [Ca2+] that differs substantially from the mean [Ca2+]. A striking example is provided by mitochondria, intracellular organelles that have been overlooked for a long time in the field of calcium signalling due to the low affinity of their Ca(2+)-uptake pathways. We will summarize here some of the evidence indicating that these organelles actively participate in Ca2+ homeostasis in physiological conditions (with consequences not only for the control of their function, but also for the modulation of the complexity of calcium signals) because they have the capability to respond to microdomains of high [Ca2+] transiently generated in their proximity by the opening of Ca2+ channels.     

Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease

Endosomes, lysosomes and lysosome-related organelles are emerging as important Ca2+ storage cellular compartments with a central role in intracellular Ca2+ signalling. Endocytosis at the plasma membrane forms endosomal vesicles which mature to late endosomes and culminate in lysosomal biogenesis. During this process, acquisition of different ion channels and transporters progressively changes the endolysosomal luminal ionic environment (e.g. pH and Ca2+) to regulate enzyme activities, membrane fusion/fission and organellar ion fluxes, and defects in these can result in disease. In the present review we focus on the physiology of the inter-related transport mechanisms of Ca2+ and H+ across endolysosomal membranes. In particular, we discuss the role of the Ca2+-mobilizing messenger NAADP (nicotinic acid adenine dinucleotide phosphate) as a major regulator of Ca2+ release from endolysosomes, and the recent discovery of an endolysosomal channel family, the TPCs (two-pore channels), as its principal intracellular targets. Recent molecular studies of endolysosomal Ca2+ physiology and its regulation by NAADP-gated TPCs are providing exciting new insights into the mechanisms of Ca2+-signal initiation that control a wide range of cellular processes and play a role in disease. These developments underscore a new central role for the endolysosomal system in cellular Ca2+ regulation and signalling.     

Local Ca2+ signals in cellular signalling

Local Ca2+ rises and propagated Ca2+ signals represent different patterns that are differentially decoded for fine tuning cellular signalling. This Ca2+ concentration plasticity is absolutely required to allow adaptation to different needs of the cells ranging from contraction or increased learning to proliferation and cell death. A wide diversity of molecular structures and specific location of Ca2+ signalling molecules confer spatial and temporal versatility to the Ca2+ changes allowing specific cellular responses to be elicited. Various types of local Ca2+ signals have been described. Ca2+ spikes correspond to Ca2+ signals spanning several micrometers but displaying limited propagation into a cell leading to regulation of cellular functions in one particular zone of this cell. This is of particular relevance in cells presenting distinct morphological specializations, i.e. apical versus basal sites or dendritic versus somatic/axonal sites. More stereotyped elementary Ca2+ events (denominated Ca2+ sparks or Ca2+ puffs depending on the type of endoplasmic reticulum Ca2+ release channel involved) are highly confined and non-propagated Ca2+ rises which are observed in the close neighbouring of the Ca2+ channels. These elementary Ca2+ events play a major role in controlling cellular excitability. Elementary Ca2+ events involve Ca2+ release channels such as the ryanodine receptors (RyRs) and the inositol 1,4,5-trisphosphate receptors (InsP3Rs). The molecular bases underlying the various local Ca2+ release events will be discussed by reviewing the channels and particularly the different isoforms of RyRs and InsP3Rs and their role in inducing localized Ca2+ responses. These calcium release events are controlled by various second messengers and are regulated by Ca2+ channel-associated proteins, intra-luminal Ca2+ content of the endoplasmic reticulum (ER) and other Ca2+ organelles. We will discuss on how the control of local cellular Ca2+ content may account for cellular functions in physiological and physiopathological conditions.     

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.     

S-glutathionylation decreases Mg2+ inhibition and S-nitrosylation enhances Ca2+ activation of RyR1 channels

We have analyzed the effects of the endogenous redoxactive agents S-nitrosoglutathione and glutathione disulfide, and the NO donor NOR-3, on calcium release kinetics mediated by ryanodine receptor channels. Incubation of triad-enriched sarcoplasmic reticulum vesicles isolated from mammalian skeletal muscle with these three agents elicits different responses. Glutathione disulfide significantly reduces the inhibitory effect of Mg2+ without altering Ca2+ activation of release kinetics, whereas NOR-3 enhances Ca2+ activation of release kinetics without altering Mg2+ inhibition. Incubation with S-nitrosoglutathione produces both effects; it significantly enhances Ca2+ activation of release kinetics and diminishes the inhibitory effect of Mg2+ on this process. Triad incubation with [35S]nitrosoglutathione at pCa 5 promoted 35S incorporation into 2.5 cysteine residues per channel monomer; this incorporation decreased significantly at pCa 9. These findings indicate that S-nitrosoglutathione supports S-glutathionylation as well as the reported S-nitrosylation of ryanodine receptor channels (Sun, J., Xu, L., Eu, J. P., Stamler, J. S., and Meissner, G. (2003) J. Biol. Chem. 278, 8184-8189). The combined results suggest that S-glutathionylation of specific cysteine residues can modulate channel inhibition by Mg2+, whereas S-nitrosylation of different cysteines can modulate the activation of the channel by Ca2+. Possible physiological and pathological implications of the activation of skeletal Ca2+ release channels by endogenous redox species are discussed.     

Calcium influx mediated by the inwardly rectifying K+ channel Kir4.1 (KCNJ10) at low external K+ concentration

COS-1 cells with heterologeous expression of the Kir4.1 (KCNJ10) channel subunit, possess functional Kir4.1 channels and become capable to generating cytosolic Ca2+ transients, upon lowering of the extracellular K+ concentration to 2 mM or below. These Ca2+ transients are blocked by external Ba2+ (100 microM). Acute brain stem slices from wild-type mice (second post-natal week), which were loaded with the fluorescent Ca2+ indicator Oregon Green BAPTA-1-AM, were exposed to 0.2 mM K+. Under these conditions astrocytes, but not neurons, responded with cytosolic Ca2+ elevations in wild-type mice. This astrocyte-specific response has previously been used to identify astroglial cells type [R. Dallwig, H. Vitten, J.W. Deitmer, A novel barium-sensitive calcium influx into rat astrocytes at low external potassium. Cell Calcium 28 (2000) 247-259]. In Kir4.1 knock-out (Kir4.1-/-) mice, the number of responding cells was dramatically reduced and the Ca2+ transients in responding cells were significantly smaller than in wild-type mice. Our results indicate that Kir4.1 channels are the molecular substrate for the observed Ca2+ influx in astrocytes under conditions of low external K+-concentration.     

Effects of dihydropyridines and inorganic calcium blockers on aggregation and on intracellular free calcium in platelets.

[Ca2+]i increase is necessary in physiological platelet activity, particularly aggregation and release. The increase of [Ca2+]i observed during platelet activation depends in part on Ca2+ influx from the extracellular medium. The participation of voltage-operated Ca2+ channels as a pathway for Ca2+ entry is controversial. In the present study we have attempted to reinvestigate this problem by measuring aggregation and [Ca2+]i changes in platelets activated by ADP or thrombin and incubated with organic or inorganic blockers of calcium channels. The main findings of the present paper can be summarized as follows: (i) Ni2+, Co2+ and Mn2+, well known inorganic blockers of Ca2+ channels, inhibited platelet aggregation induced by ADP or thrombin in a dose-dependent manner, Ni2+ being the most effective agent. (ii) Thrombin induced a rise in free [Ca2+]i in platelets incubated both in 1 mmol/l Ca(2+)-containing medium and in nominally Ca(2+)-free medium; the rise of free [Ca2+]i was in the first case up to 370 +/- 31 nmol/l and in the second case up to 242 +/- 26 nmol/l, indicating that this observed difference was due to Ca2+ entry from the extracellular medium. Co2+ and Ni2+ abolished that difference by inhibiting Ca2+ influx. (iii) Nisoldipine, nitrendipine and nimodipine (10-50 nmol/l) inhibited in a dose-dependent manner platelet aggregation induced by either ADP or thrombin in platelets incubated in normal-Ca2+ normal-K+ medium, also, aggregation was inhibited to a similar extent in platelets incubated in normal-Ca2+ high-K+ medium. (iv) Nisoldipine--the most effective dihydropyridine to inhibit platelet aggregation--also inhibited Ca2+ influx in platelets incubated in normal-Ca2+ medium, either in normal-K+ or high-K+ media. Our data support the existence of voltage-operated, dihydropyridine-sensitive calcium channels (L-type) and a physiological role for them in platelet function.     

2-Aminoethyl diphenylborinate analogues: selective inhibition for store-operated Ca2+ entry

Capacitative calcium entry (CCE), the mechanism that replenishes the internal Ca2+ stores with Ca2+ from the extracellular milieu in response to depletion of the store, is mediated by Ca2+ channels in the plasma membrane generally referred to as store-operated channels (SOCs). However, the roles of SOCs in the more physiological context have been fully elucidated. 2-Aminoethyl diphenylborinate (2-APB) strongly inhibits SOCs, as well as inositol-1,4,5 trisphosphate (IP3) receptors. In the present study, we screened a library of 166 2-APB analogues for effects on CCE and IP3-induced Ca2+ release in order to discover specific SOC inhibitors, and found that some blocked both store-operated and receptor-operated Ca2+ influx more strongly and selectively than 2-APB. Indeed, these new compounds ceased the prolonged intracellular Ca2+ oscillations induced by a low concentration of ATP in CHO-K1 cells. These novel SOC inhibitors will be valuable pharmacological and biochemical tools for elucidating the physiological roles.     

L-type Ca2+ channels in Ca2+ channelopathies

Voltage-gated L-type Ca2+ channels (LTCCs) mediate depolarization-induced Ca2+ entry in electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells. In this review we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within pore-forming alpha1 subunits causing incomplete congenital stationary night blindness, malignant hyperthermia sensitivity or hypokalemic periodic paralysis. However, studies in mice revealed that LTCC dysfunction also contributes to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting molecular tools to elucidate the contribution of different LTCC isoforms to human diseases.     

Domain model for Ca2(+)-inactivation of Ca2+ channels at low channel density.

The "shell" model for Ca2(+)-inactivation of Ca2+ channels is based on the accumulation of Ca2+ in a macroscopic shell beneath the plasma membrane. The shell is filled by Ca2+ entering through open channels, with the elevated Ca2+ concentration inactivating both open and closed channels at a rate determined by how fast the shell is filled. In cells with low channel density, the high concentration Ca2+ "shell" degenerates into a collection of nonoverlapping "domains" localized near open channels. These domains form rapidly when channels open and disappear rapidly when channels close. We use this idea to develop a "domain" model for Ca2(+)-inactivation of Ca2+ channels. In this model the kinetics of formation of an inactivated state resulting from Ca2+ binding to open channels determines the inactivation rate, a mechanism identical with that which explains single-channel recordings on rabbit-mesenteric artery Ca2+ channels (Huang Y., J. M. Quayle, J. F. Worley, N. B. Standen, and M. T. Nelson. 1989. Biophys. J. 56:1023-1028). We show that the model correctly predicts five important features of the whole-cell Ca2(+)-inactivation for mouse pancreatic beta-cells (Plants, T. D. 1988. J. Physiol. 404:731-747) and that Ca2(+)-inactivation has only minor effects on the bursting electrical activity of these cells.