Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels

Acute modulation of P/Q-type (alpha1A) calcium channels by neuronal activity-dependent changes in intracellular Ca2+ concentration may contribute to short-term synaptic plasticity, potentially enriching the neurocomputational capabilities of the brain. An unconventional mechanism for such channel modulation has been proposed in which calmodulin (CaM) may exert two opposing effects on individual channels, initially promoting ('facilitation') and then inhibiting ('inactivation') channel opening. Here we report that such dual regulation arises from surprising Ca2+-transduction capabilities of CaM. First, although facilitation and inactivation are two competing processes, both require Ca2+-CaM binding to a single 'IQ-like' domain on the carboxy tail of alpha1A; a previously identified 'CBD' CaM-binding site has no detectable role. Second, expression of a CaM mutant with impairment of all four of its Ca2+-binding sites (CaM1234) eliminates both forms of modulation. This result confirms that CaM is the Ca2+ sensor for channel regulation, and indicates that CaM may associate with the channel even before local Ca2+ concentration rises. Finally, the bifunctional capability of CaM arises from bifurcation of Ca2+ signalling by the lobes of CaM: Ca2+ binding to the amino-terminal lobe selectively initiates channel inactivation, whereas Ca2+ sensing by the carboxy-terminal lobe induces facilitation. Such lobe-specific detection provides a compact means to decode local Ca2+ signals in two ways, and to separately initiate distinct actions on a single molecular complex.     

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.     

Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the beta-subunit. The reduced channel activities are due to a decrease in alpha1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with beta-subunits, regulates Ca2+ channel expression at the cell surface.     

A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels

Ca2+/calmodulin-dependent regulation of voltage-gated CaV1-2 Ca2+ channels shows extraordinary modes of spatial Ca2+ decoding and channel modulation, vital for many biological functions. A single calmodulin (CaM) molecule associates constitutively with the channel's carboxy-terminal tail, and Ca2+ binding to the C-terminal and N-terminal lobes of CaM can each induce distinct channel regulations. As expected from close channel proximity, the C-lobe responds to the roughly 100-microM Ca2+ pulses driven by the associated channel, a behaviour defined as 'local Ca2+ selectivity'. Conversely, all previous observations have indicated that the N-lobe somehow senses the far weaker signals from distant Ca2+ sources. This 'global Ca2+ selectivity' satisfies a general signalling requirement, enabling a resident molecule to remotely sense cellular Ca2+ activity, which would otherwise be overshadowed by Ca2+ entry through the host channel. Here we show that the spatial Ca2+ selectivity of N-lobe CaM regulation is not invariably global but can be switched by a novel Ca2+/CaM-binding site within the amino terminus of channels (NSCaTE, for N-terminal spatial Ca2+ transforming element). Native CaV2.2 channels lack this element and show N-lobe regulation with a global selectivity. On the introduction of NSCaTE into these channels, spatial Ca2+ selectivity transforms from a global to local profile. Given this effect, we examined CaV1.2/CaV1.3 channels, which naturally contain NSCaTE, and found that their N-lobe selectivity is indeed local. Disruption of this element produces a global selectivity, confirming the native function of NSCaTE. Thus, differences in spatial selectivity between advanced CaV1 and CaV2 channel isoforms are explained by the presence or absence of NSCaTE. Beyond functional effects, the position of NSCaTE on the channel's amino terminus indicates that CaM can bridge the amino terminus and carboxy terminus of channels. Finally, the modularity of NSCaTE offers practical means for understanding the basis of global Ca2+ selectivity.     

Involvement of Ca<Superscript>2+</Superscript>/CaM in the signal transduction of acetylcholine regulating stomatal movement

It has been known that the neurotransmitter acetylcholine (ACh) also exists in plants and is able to regulate the movement of stomata.In another aspect,Ca^2 /CaM as the second messengers have a critical role of signal transduction in stomatal guard-cell,Here we showed that Ca^2 /CaM were also involved in the ACh regulated stomatal movement,In the medium containing Ca^2 ,the Ca^2 channel blockers (NIF and Ver)and CaM inhibitors (TFP and W7 ) could neutralize the ACh induced stomatal opening,however,they are ineffective in the medium containing K^ ,Those results indicated that Ca^2 /CaM were involved in the signal transduction pathway of ACh regulating stomatal movement.     

Charge movement during Na+ translocation by native and cloned cardiac Na+/Ca2+ exchanger

Na+/Ca2+ exchange is electrogenic and moves one net positive charge per cycle. Although the cardiac exchanger has a three-to-one Na+/Ca2+ stoichiometry, details of the reaction cycle are not well defined. Here we associate Na+ translocation by the cardiac exchanger with positive charge movement in giant membrane patches from cardiac myocytes and oocytes expressing the cloned cardiac Na+/Ca2+ exchanger. The charge movements are initiated by step increments of the cytoplasmic Na+ concentration in the absence of Ca2+. Giant patches from control oocytes lack both steady-state Na+/Ca2+ exchange current (INaCa) and Na(+)-induced charge movements. Charge movements indicate about 400 exchangers per micron 2 in guinea-pig sarcolemma. Fully activated INaCa densities (20-30 microA cm-2) indicate maximum turnover rates of 5,000 s-1. As has been predicted for consecutive exchange models, the apparent ion affinities of steady state INaCa increase as the counterion concentrations are decreased. Consistent with an electroneutral Ca2+ translocation, we find that voltage dependence of INaCa in both directions is lost as Ca2+ concentration is decreased. The principal electrogenic step seems to be at the extracellular end of the Na+ translocation pathway.     

Identification of Na-Ca exchange current in single cardiac myocytes

In cardiac muscle the exchange of intracellular Ca2+ for extracellular Na+ is an important transport mechanism for regulation of the intracellular free Ca2+ concentration [( Ca]i) and hence the contractile strength of the heart. Due to its stoichiometry of greater than or equal to 3:1 Na+/Ca2+ (refs 3,5), Na-Ca exchange is supposed to generate a current across the cell membrane. It is thought that such a current may contribute to cardiac action potential and physiological or pathological pacemaker activity. Although the occurrence of Na-Ca exchange is well documented, a membrane current generated by this transport has not been identified unequivocally. Previous attempts to detect such a current in multicellular preparations, for example, by measuring small current differences after varying the extracellular ionic composition, although providing evidence, did not rule out other possible interpretations. Here we demonstrate that a transient rise in [Ca]i caused by release of Ca from sarcoplasmic reticulum (SR) generates a membrane current in cardiac myocytes. The dependence of this current on the transmembrane gradients for Na+ and Ca2+ and on membrane potential meets the criteria for a current produced by electrogenic Na-Ca exchange. Cyclic activation of this current by release of Ca from the SR can cause maintained spontaneous activity, suggesting that Na-Ca exchange contributes to certain forms of cardiac pacemaking.     

TRIC channels are essential for Ca2+ handling in intracellular stores

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.     

Calmodulin modulation of ion channels and receptors

Ion channels and receptors are the structural basis for neural signaling and transmission. Recently, the function of ion channels and receptors has been demonstrated to be modulated by many intracellular and extracellular chemicals and signaling molecules. Increasing evidence indicates that the complexity and plasticity of the function of central nervous system is determined by the modulation of ion channels and receptors. Among various mechanisms, Ca 2+ signaling pathways play important roles in neuronal activity and some pathological changes. Ca 2+ influx through ion channels and receptors can modulate its further influx in a feedback way or modulate other ion channels and receptors. The common feature of the modulation is that Ca 2+ /calmodulin (CaM) is the universal mediator. CaM maintains the coordination among ion channels/receptors and intracellular Ca 2+ homeostasis by feedback modulation of ion channels/receptors activity. This review focuses on the modulating processes of ion channels and receptors mediated by CaM, and further elucidates the mechanisms of Ca 2+ signaling.     

Calmodulin modulation of ion channels and receptors?

：Ion channels and receptors are the structural basis for neural signaling and transmission. Recently, the function of ion channels and receptors has been demonstrated to be modulated by many intracellular and extracellular chemicals and signaling molecules. Increasing evidence indicates that the complexity and plasticity of the function of central nervous system is determined by the modulation of ion channels and receptors. Among various mechanisms, Ca 2+ signaling pathways play important roles in neuronal activity and some pathological changes. Ca 2+ influx through ion channels and receptors can modulate its further influx in a feedback way or modulate other ion channels and receptors. The common feature of the modulation is that Ca 2+ /calmodulin (CaM) is the universal mediator. CaM maintains the coordination among ion channels/receptors and intracellular Ca 2+ homeostasis by feedback modulation of ion channels/receptors activity. This review focuses on the modulating processes of ion channels and receptors mediated by CaM, and further elucidates the mechanisms of Ca 2+ signaling.     

Repeat I of the dihydropyridine receptor is critical in determining calcium channel activation kinetics.

Membrane depolarization causes many kinds of ion channels to open, a process termed activation. For both Na+ channels and Ca2+ channels, kinetic analysis of current has suggested that during activation the channel undergoes several conformational changes before reaching the open state. Structurally, these channels share a common motif: the central element is a large polypeptide with four repeating units of homology (repeats I-IV), each containing a voltage-sensing region, the S4 segment. This suggests that the distinct conformational transitions inferred from kinetic analysis may be equated with conformational changes of the individual structural repeats. To investigate the molecular basis of channel activation, we constructed complementary DNAs encoding chimaeric Ca2+ channels in which one or more of the four repeats of the skeletal muscle dihydropyridine receptor are replaced by the corresponding repeats derived from the cardiac dihydropyridine receptor. We report here that repeat I determines whether the chimaeric Ca2+ channel shows slow (skeletal muscle-like) or rapid (cardiac-like) activation.     

Multiple transport modes of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger (NCX1; ref. 2) is a bi-directional Ca2+ transporter that contributes to the electrical activity of the heart. When, and if, Ca2+ is exported or imported depends on the Na+/Ca2+ exchange ratio. Whereas a ratio of 3:1 (Na+:Ca2+) has been indicated by Ca2+ flux equilibrium studies, a ratio closer to 4:1 has been indicated by exchange current reversal potentials. Here we show, using an ion-selective electrode technique to quantify ion fluxes in giant patches, that ion flux ratios are approximately 3.2 for maximal transport in either direction. With Na+ and Ca2+ on both sides of the membrane, net current and Ca2+ flux can reverse at different membrane potentials, and inward current can be generated in the absence of cytoplasmic Ca2+, but not Na+. We propose that NCX1 can transport not only 1 Ca2+ or 3 Na+ ions, but also 1 Ca2+ with 1 Na+ ion at a low rate. Therefore, in addition to the major 3:1 transport mode, import of 1 Na+ with 1 Ca2+ defines a Na+-conducting mode that exports 1 Ca2+, and an electroneutral Ca2+ influx mode that exports 3 Na+. The two minor transport modes can potentially determine resting free Ca2+ and background inward current in heart.     

Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.     

Na-Ca exchange current in mammalian heart cells

Electrogenic Na-Ca exchange has been known to act in the cardiac sarcolemma as a major mechanism for extruding Ca ions. Ionic flux measurements in cardiac vesicles have recently suggested that the exchange ratio is probably 3 Na:1 Ca, although a membrane current generated by such a process has not been isolated. Using the intracellular perfusion technique combined with the whole-cell voltage clamp, we were able to load Na+ inside and Ca2+ outside the single ventricular cells of the guinea pig and have succeeded in recording an outward Na-Ca exchange current while blocking most other membrane currents. The current is voltage-dependent, blocked by La3+ and does not develop in the absence of intracellular free Ca2+. This report presents the first direct measurement of the cardiac Na-Ca exchange current, and should facilitate the study of Ca2+ fluxes during cardiac activity, together with various electrical changes attributable to the Na-Ca exchange and the testing of proposed models.     

CaT1 manifests the pore properties of the calcium-release-activated calcium channel

The calcium-release-activated Ca2+channel, ICRAC, is a highly Ca2+-selective ion channel that is activated on depletion of either intracellular Ca2+ levels or intracellular Ca2+ stores. The unique gating of ICRAC has made it a favourite target of investigation for new signal transduction mechanisms; however, without molecular identification of the channel protein, such studies have been inconclusive. Here we show that the protein CaT1 (ref. 4), which has six membrane-spanning domains, exhibits the unique biophysical properties of ICRAC when expressed in mammalian cells. Like ICRAC, expressed CaT1 protein is Ca2+ selective, activated by a reduction in intracellular Ca2+ concentration, and inactivated by higher intracellular concentrations of Ca2+. The channel is indistinguishable from ICRAC in the following features: sequence of selectivity to divalent cations; an anomalous mole fraction effect; whole-cell current kinetics; block by lanthanum; loss of selectivity in the absence of divalent cations; and single-channel conductance to Na+ in divalent-ion-free conditions. CaT1 is activated by both passive and active depletion of calcium stores. We propose that CaT1 comprises all or part of the ICRAC pore.     

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.     

Cholecystokinin induces a decrease in Ca2+ current in snail neurons that appears to be mediated by protein kinase C

Three distinct classes of protein kinases have been shown to regulate Ca2+ current in excitable tissues. Cyclic AMP-dependent protein kinase mediates the action of noradrenaline on the Ca2+ current of cardiac muscle cells. Cyclic GMP-dependent protein kinase mediates the serotonin-induced modulation of the Ca2+ current in identified snail neurons. The Ca2+/diacylglycerol-dependent protein kinase (protein kinase C) has also been found to regulate Ca2+ currents of neurons. However, no neurotransmitter has yet been shown to regulate Ca2+ current through the activation of protein kinase C. We now report that cholecystokinin, a widely occurring neuropeptide which is present in molluscan neuron, modulates the Ca2+ current in identified neurons of the snail Helix aspersa, and that this effect appears to be mediated by protein kinase C. Specifically, sulphated cholecystokinin octapeptide 26-33 (CCK8), activators of protein kinase C, and intracellular injection of protein kinase C, all shorten the Ca2+-dependent action potential and decrease the amplitude of the Ca2+ current in these cells. All these effects are not reversible within the duration of the experiments. Moreover, intracellular injections of low concentrations of protein kinase C, which are ineffective by themselves, enhance the effectiveness of low concentrations of CCK8 on the Ca2+ current.     

Activation of sodium-proton exchange is a prerequisite for Ca2+ mobilization in human platelets

Stimulated platelets take up sodium ions and release hydrogen ions due to activation of Na+/H+ exchange resulting in cytoplasmic alkalinization. Suppression of Na+/H+ exchange either by removal of extracellular Na+ or by application of amiloride inhibits shape change, secretion of granule contents and aggregation. The data we present here indicate that inhibition of this transport by ethylisopropyl-amiloride or by lowering extracellular sodium reduces or even completely suppresses the rise in cytoplasmic free Ca2+ concentration that is essential for platelet aggregation in response to thrombin. We also demonstrate that cytoplasmic alkalinization produced by exposure to the ionophore monensin sensitizes the human platelet response to stimulation by thrombin resulting in enhanced Ca2+ mobilization and aggregability. We conclude that an increase in intracellular pH evoked by activation of Na+/H+ counter transport is an important signal in stimulus-response coupling and forms an essential step in the cascade of events required to increase cytoplasmic free Ca2+ in platelets.     

Rotational movement during cyclic nucleotide-gated channel opening

Cyclic nucleotide-gated (CNG) channels are crucial components of visual, olfactory and gustatory signalling pathways. They open in response to direct binding of intracellular cyclic nucleotides and thus contribute to cellular control of both the membrane potential and intracellular Ca2+ levels. Cytosolic Ni2+ potentiates the rod channel (CNG1) response to cyclic nucleotides and inhibits the olfactory channel (CNG2) response. Modulation is due to coordination of Ni2+ by channel-specific histidines in the C-linker, between the S6 transmembrane segment and the cyclic nucleotide-binding domain. Here we report, using a histidine scan of the initial C-linker of the CNG1 channel, stripes of sites producing Ni2+ potentiation or Ni2+ inhibition, separated by 50 degrees on an alpha-helix. These results suggest a model for channel gating where rotation of the post-S6 region around the channel's central axis realigns the Ni2+-coordinating residues of multiple subunits. This rotation probably initiates movement of the S6 and pore opening.