Ca2+-dependent and Ca2+-independent phagocytosis in human neutrophils

The phagocytic function of neutrophils is a crucial element in host defence against invading microorganisms. Two main specific receptor-mediated mechanisms operate in the phagocyte plasma membrane, one recognizing the C3b/bi fragment of complement and the other the Fc domain of immunoglobulin G (ref. 1). There is evidence that phagocytosis mediated by these receptors differs in the number and nature of the intracellular signals generated. However, the mechanisms by which receptor binding is transduced into a signal that generates the formation of the phagocyte pseudopod is not known, although extensive biochemical evidence has allowed the postulate that calcium ion gradients in the peripheral cytoplasm, by interacting with calcium-sensitive contractile proteins, initiate the process of engulfment. Using the high-affinity fluorescent calcium indicator quin2 both to measure and to buffer intracellular calcium ([Ca2+]i), we show here that in human neutrophils two mechanisms of phagocytosis coexist: a [Ca2+]i-dependent and modulated phagocytosis, triggered by activation of the Fc receptor, and a [Ca2+]i-independent mechanism triggered by the activation of the C3b/bl receptors.     

Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel

High-conductance voltage- and Ca(2+)-activated K(+) channels function in many physiological processes that link cell membrane voltage and intracellular Ca(2+) concentration, including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning. Like other voltage-dependent K(+) channels, Ca(2+)-activated K(+) channels open when the cell membrane depolarizes, but in contrast to other voltage-dependent K(+) channels, they also open when intracellular Ca(2+) concentrations rise. Channel opening by Ca(2+) is made possible by a structure called the gating ring, which is located in the cytoplasm. Recent structural studies have defined the Ca(2+)-free, closed, conformation of the gating ring, but the Ca(2+)-bound, open, conformation is not yet known. Here we present the Ca(2+)-bound conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of Ca(2+), opens like the petals of a flower. The degree to which it opens explains how Ca(2+) binding can open the transmembrane pore. These findings present a molecular basis for Ca(2+) activation of K(+) channels and suggest new possibilities for targeting the gating ring to treat conditions such as asthma and hypertension.     

Heat and hyposmotic stimulation increase in [Ca^2＋]i by Ca^2＋ influx in rat synoviocytes

Rheumatoid arthritis （RA）, which is marked by inflammatory synovitis, is a common, chronic autoimmune-disease, whose pathogenesis is complex and still unclear. In order to explore the effects of heat and hyposmotic stimuli on synoviocytes in rheumatoid arthritis, the changes of [Ca^2＋]i induced by heat, hyposmotic and 4α-PDD stimuli were observed in synoviocytes. [Ca^2＋]i elevation induced by heat 28℃, hyposmotic and 4α-PDD stimuli is found to be positively relative to increasing temperature, decreasing osmolality and rising concentration of 4α-PDD. Results show that there is reciprocity among these stimuli and desensitization, and that [Ca^2＋]i elevation depends on Ca^2＋ influx, but not necessarily links to Ca^2＋ release from intracellular stores and voltage-dependent Ca^2＋ channel in synoviocytes. The above characteristics of Ca^2＋ influx are similar to those of TRPV4. A probable mechanism has been suggested that heat and hyposmotic stimulation might increase the level of [Ca^2＋]i by activating the TRPV4-like channel and Ca^2＋ influx in the synoviocytes.     

The large-conductance Ca2+-activated K+ channel is essential for innate immunity

Neutrophil leukocytes have a pivotal function in innate immunity. Dogma dictates that the lethal blow is delivered to microbes by reactive oxygen species (ROS) and halogens, products of the NADPH oxidase, whose impairment causes immunodeficiency. However, recent evidence indicates that the microbes might be killed by proteases, activated by the oxidase through the generation of a hypertonic, K+-rich and alkaline environment in the phagocytic vacuole. Here we show that K+ crosses the membrane through large-conductance Ca2+-activated K+ (BK(Ca)) channels. Specific inhibitors of these channels, iberiotoxin and paxilline, blocked oxidase-induced 86Rb+ fluxes and alkalinization of the phagocytic vacuole, whereas NS1619, a BK(Ca) channel opener, enhanced both. Characteristic outwardly rectifying K+ currents, reversibly inhibited by iberiotoxin, were demonstrated in neutrophils and eosinophils and the expression of the alpha-subunit of the BK channel was confirmed by western blotting. The channels were opened by the combination of membrane depolarization and elevated Ca2+ concentration, both consequences of oxidase activity. Remarkably, microbial killing and digestion were abolished when the BK(Ca) channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process.     

Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels

Animal cells initially swell in hypotonic media by osmotic water equilibration, but their volume is subsequently regulated by a net loss of KCl and amino acids with concomitant loss of cell water. Mechanisms for regulating cell volume are important in allowing cells to adapt to variations in external tonicity and metabolic load. In red cells the KCl loss is mediated by electroneutral ion transport mechanisms. In contrast, conductive K+ and Cl- transport pathways are activated during regulatory volume decrease in several cell types including epithelia. The activation seems to be mediated by internal Ca2+, but the detailed mechanism is not known. In a leaky epithelium, the choroid plexus epithelium, we have found a cation-selective, Ca2+-permeable channel which opens with membrane stretch. The epithelium also contains a high density of the large (approximately 200 pS) type of Ca2+- voltage-activated K+ channel. Both channels are normally closed. I propose that in hypotonic media, the stretching of the cell membrane produced by the initial swelling causes influx of Ca2+ through the stretch-activated channels, which activates the neighbouring large K+ channels to produce increased K+ outflux with associated loss of cell water.     

Effects of La3+ and Gd3+ on Ca2+ influx in rat hepatoma H-35 cells

Effects of La³⁺ and Gd³⁺ on Ca²⁺ influx were investigated in rat hepatoma H-35 cells by measuring the initial rate of⁴⁵Ca²⁺ uptake. It was found that the maximum initial rate of Ca²⁺ uptake was increased six-to ten-fold at low concentrations of La³⁺ and Gd³⁺. Kinetic analyses by measuring the initial rate of Ca²⁺ influx at different external Ca²⁺ concentrations indicated the existence of two intracellular exchangeable components in the basal Ca²⁺ system, with low and high affinities for Ca²⁺, and only one class of Ca²⁺ binding sites was observed in the La³⁺-or Gd³⁺-treated cells. For high affinity, La³⁺ and Gd³⁺ increased both kinetic parametersK _m andV _max of basai Ca²⁺ influx. La³⁺ and Gd³⁺ compete directly with Ca²⁺ for Ca²⁺ binding site for low affinity. The kinetics is competitive.     

Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin

Small-conductance Ca2+-activated K+ channels (SK channels) are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are heteromeric complexes that comprise pore-forming alpha-subunits and the Ca2+-binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM-binding domain (CaMBD), which is located in an intracellular region of the alpha-subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. Here we report the 1.60 A crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three alpha-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.     

The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement

Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants.     

Ca2+ channel modulation by 8-bromocyclic AMP in cultured heart cells

Modulation of ion channels is of increasing interest as it is an important step in the regulation of cellular functions. We have analysed the effect of 8-bromocyclic AMP on Ca2+ channels in cultured cardiac cells by the patch-clamp method and report here that there was a large increase in the probability of opening of the channels. On the basis of a recently proposed kinetic reaction scheme we suggest that cyclic AMP-dependent phosphorylation of Ca2+ channels primarily promotes the forward rate constants which lead to the open state of a Ca2+ channel during depolarization.     

Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2+ channel

In mammals, sperm cells become motile during ejaculation and swim up the female reproductive tract. Before fertilization and to overcome various barriers, their motility must be hyperactivated, a motion that is characterized by vigorous asymmetric tail beating. Hyperactivation requires an increase in calcium in the flagella, a process that probably involves plasmalemmal ion channels. Numerous attempts in the past two decades to understand sperm cell channels have been frustrated by the difficulty of measuring spermatozoan transmembrane ion currents. Here, by using a simple approach to patch-clamp spermatozoa and to characterize whole-spermatozoan currents, we describe a constitutively active flagellar calcium channel that is strongly potentiated by intracellular alkalinization. This current is not present in spermatozoa lacking the sperm-specific putative ion channel protein, CatSper1. This plasma membrane protein of the six transmembrane-spanning ion channel superfamily is specifically localized to the principal piece of the sperm tail and is required for sperm cell hyperactivation and male fertility. Our results identify CatSper1 as a component of the key flagellar calcium channel, and suggest that intracellular alkalinization potentiates CatSper current to increase intraflagellar calcium and induce sperm hyperactivation.     

A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle

Receptor-operated Ca2+ entry has been proposed as a signalling mechanism in many cells. Receptor-operated Ca2+ channels (ROCs) were first postulated in smooth muscle by Bolton, van Breemen and Somlyo and Somlyo, but recordings of directly ligand-gated Ca2+ current are lacking. Here we describe receptor-operated Ca2+ current evoked in arterial smooth muscle cells by ATP, a sympathetic neurotransmitter. ATP activates channels with approximately 3:1 selectivity for Ca2+ over Na+ at near-physiological concentrations and with a unitary conductance of approximately 5 pS in 110 mM Ca2+ or Ba2+. The channels can be opened even at very negative potentials and resist inhibition by cadmium or nifedipine, unlike voltage-gated Ca2+ channels; they are not blocked by Mg2+, unlike NMDA (N-methyl-D-aspartate)-activated channels; they are directly activated by ligand, without involvement of readily diffusible second messengers, unlike cation channels in neutrophils and T lymphocytes. Thus, the ATP-activated channels provide a distinct mechanism for excitatory synaptic current and Ca2+ entry in smooth muscle.     

Metabolic stabilization of endplate acetylcholine receptors regulated by Ca2+ influx associated with muscle activity.

During formation of the neuromuscular junction, acetylcholine receptors in the endplate membrane become metabolically stabilized under neural control, their half-life increasing from about 1 day to about 10 days. The metabolic stability of the receptors is regulated by the electrical activity induced in the muscle by innervation. We report here that metabolic stabilization of endplate receptors but not of extrajunctional receptors can be induced in the absence of muscle activity if muscles are treated with the calcium ionophore A23187. Acetylcholine receptor stabilization was also induced by culturing non-stimulated muscle in elevated K+ with the Ca2+ channel activator (+)-SDZ202-791. Conversely, activity-dependent receptor stabilization is prevented in muscle stimulated in the presence of the Ca2+ channel blockers (+)-PN200-110 or D-600. Treatment of muscles with ryanodine, which induces Ca2+ release from the sarcoplasmic reticulum in the absence of activity, does not cause stabilization of junctional receptors. Evidently, muscle activity induces metabolic acetylcholine receptor stabilization by way of an influx of Ca2+ ions through dihydropyridine-sensitive Ca2+ channels in the endplate membrane, whereas Ca2+ released from the sarcoplasmic reticulum is ineffective in this developmental process.     

Voltage and Ca2+-activated K+ channel in baso-lateral acinar cell membranes of mammalian salivary glands

Nervous or hormonal stimulation of many exocrine glands evokes release of cellular K+ (ref. 1), as originally demonstrated in mammalian salivary glands2,3, and is associated with a marked increase in membrane conductance1,4,5. We now demonstrate directly, by using the patch-clamp technique6, the existence of a K+ channel with a large conductance localized in the baso-lateral plasma membranes of mouse and rat salivary gland acinar cells. The K+ channel has a conductance of approximately 250 pS in the presence of high K+ solutions on both sides of the membrane. Although mammalian exocrine glands are believed not to possess voltage-activated channels1,7, the probability of opening the salivary gland K+ channel was increased by membrane depolarization. The frequency of channel opening, particularly at higher membrane potentials, was increased markedly by elevating the internal ionized Ca2+ concentration, as previously shown for high-conductance K+ channels from cells of neural origin8-10. The Ca2+ and voltage-activated K+ channel explains the marked cellular K+ release that is characteristically observed when salivary glands are stimulated to secrete.     

Ca2+ signalling between single L-type Ca2+ channels and ryanodine receptors in heart cells

Ca2+-induced Ca2+ release is a general mechanism that most cells use to amplify Ca2+ signals. In heart cells, this mechanism is operated between voltage-gated L-type Ca2+ channels (LCCs) in the plasma membrane and Ca2+ release channels, commonly known as ryanodine receptors, in the sarcoplasmic reticulum. The Ca2+ influx through LCCs traverses a cleft of roughly 12 nm formed by the cell surface and the sarcoplasmic reticulum membrane, and activates adjacent ryanodine receptors to release Ca2+ in the form of Ca2+ sparks. Here we determine the kinetics, fidelity and stoichiometry of coupling between LCCs and ryanodine receptors. We show that the local Ca2+ signal produced by a single opening of an LCC, named a 'Ca2+ sparklet', can trigger about 4-6 ryanodine receptors to generate a Ca2+ spark. The coupling between LCCs and ryanodine receptors is stochastic, as judged by the exponential distribution of the coupling latency. The fraction of sparklets that successfully triggers a spark is less than unity and declines in a use-dependent manner. This optical analysis of single-channel communication affords a powerful means for elucidating Ca2+-signalling mechanisms at the molecular level.     

Ca2+-activated ATPase and ATP-dependent calmodulin-stimulated Ca2+ transport in islet cell plasma membrane

Calcium is known to play an essential part in the regulation of insulin secretion in the pancreatic beta cell. Calcium influx/efflux studies indicate that glucose promotes an accumulation of calcium by the beta cell. However, interpretation of such data is particularly difficult due to the complex compartmentalization of calcium within the cell. Although indirect evidence using chlorotetracycline suggests that control of calcium homeostasis at the plasma membrane may be central to insulin secretion, the mechanism by which secretagogues influence the handling of calcium remains unknown. Despite its continuous diffusive entry, intracellular calcium is maintained in the submicromolar range by energy-dependent mechanisms. One such process which has been well characterized in erythrocytes is a plasma membrane calcium extrusion pump whose enzymatic basis is a high affinity (Ca+2 + Mg+2)ATPase. A similar mechanism regulated by insulin has recently been identified in adipocyte plasma membranes. We report here the presence of a high affinity (Ca+2 + Mg+2)ATPase and ATP-dependent calmodulin-stimulated calcium transport system in rat pancreatic islet cell plasma membranes.