High-affinity Ca2 Research Articles

Voltage Sensor Deactivation Inhibits BK Channel Opening by Mg 2+

Functional and structural studies suggest that intracellular Mg2+ activates BK channels through interaction with the voltage-sensing domain (Yang et al. 2007, 2008; Horrigan & Ma 2008; Yuan et al. 2010; Wu et al. 2010). To further explore the mechanism of activation of BK channels by Mg2+ through the low affinity E374/E399 Mg2+ sites located beneath the voltage sensors, we use single-channel analysis to study BK channels mutated to remove the high affinity Ca2+ sites. We find that 10 mM Mg2+ shortens the latency to first channel opening after a voltage jump to +100 mV from −100 mV, consistent with the hypothesis that Mg2+ can bind to the closed channel and shorten the latency. However, it is not clear whether the closed-channel binding occurs when the voltage sensors are deactivated (down) or activated (up). We therefore recorded single-channel activity in macro-patches held at constant −50 mV, where voltage sensors occasionally activate. Under this condition, 10 mM Mg2+ decreases mean closed duration and increases mean open duration. These effects are attenuated at −100 mV, and become negligible at −150 mV, where the voltage sensors are mainly deactivated. For BK channels modified to have deactivated voltage-sensors (R167E), 10 mM Mg2+ has little effect on mean closed and open durations at −50 mV. In contrast, for BK channels modified to have constitutively activated voltage sensors (R210C), 10 mM Mg2+ shortens the mean closed durations and lengthens the mean open durations at −200 mV. The above observations are consistent with a model in which voltage sensor deactivation inhibits BK channel opening by Mg2+. Supported by NIH grant AR32805 and AHA 10POST4490012.

Fundamental role of microvilli in the main functions of differentiated cells: Outline of an universal regulating and signaling system at the cell periphery

Until now, the general importance of microvilli present on the surface of almost all differentiated cells has been strongly underestimated and essential functions of these abundant surface organelles remained unrecognized. Commonly, the role of microvilli has been reduced to their putative function of cell-surface enlargement. In spite of a large body of detailed knowledge about the specific functions of microvilli in sensory receptor cells for sound, light, and odor perception, their functional importance for regulation of basic cell functions remained obscure. Here, a number of microvillar mechanisms involved in fundamental cell functions are discussed. Two structural features enable the extensive functional competence of microvilli: First, the exclusive location of almost all functional important membrane proteins on microvilli of differentiated cells and second, the function of the F-actin-based cytoskeletal core of microvilli as a structural diffusion barrier modulating the flow of low molecular substrates and ions into and out of the cell. The specific localization on microvilli of important functional membrane proteins such as glucose transporters, ion channels, ion pumps, and ion exchangers indicate the importance and diversity of microvillar functions. In this review, the microvillar mechanisms of audioreceptor, photoreceptor, and olfactory/taste receptor cells are discussed as highly specialized adaptations of a general type of microvillar mechanisms involved in regulation of important basic cell functions such as glucose transport/energy metabolism, ion channel regulation, generation and modulation of the membrane potential, volume regulation, and Ca signaling. Even the constitutive cellular defence against cytotoxic compounds, also called "multidrug resistance (MDR)," is discussed as a microvillar mechanism. A comprehensive examination of the specific properties of "cable-like" ion conduction along the microvillar core structure of F-actin allows the proposal that microvilli are specifically designed for using ionic currents as cellular signals. In view of the multifaceted gating and signaling properties of TRP channels, the possible role of microvilli as a universal gating device for TRP channel regulation is discussed. Combined with the role of the microvillar core bundle of actin filaments as high-affinity Ca store, microvilli may turn out as highly specialized Ca signaling organelle involved in store-operated Ca entry (SOCE) and initiation of nonlinear Ca signals such as waves and oscillations.

Characterization of a dockerin‐based affinity tag: application for purification of a broad variety of target proteins

Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high-affinity Ca(2+)-dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (K(A)) of 7.7 × 10(8)M(-1), calculated by a competitive enzyme-linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum β-glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody-binding ZZ-domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T-6 as a model system. In addition, the enzymatic activity of a C. thermocellum β-glucosidase, purified using this approach, was tested and found to be similar to that of a β-glucosidase preparation (without the ΔDoc tag) purified using the standard His-tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose-based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca(2+)-binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification.

Exocytosis, dependent on Ca2+ release from Ca2+ stores, is regulated by Ca2+ microdomains

The relationship between the cellular Ca2+ signal and secretory vesicle fusion (exocytosis) is a key determinant of the regulation of the kinetics and magnitude of the secretory response. Here, we have investigated secretion in cells where the exocytic response is controlled by Ca2+ release from intracellular Ca2+ stores. Using live-cell two-photon microscopy that simultaneously records Ca2+signals and exocytic responses, we provide evidence that secretion is controlled by changes in Ca2+ concentration [Ca2+] in relatively large-volume microdomains. Our evidence includes: (1) long latencies (>2 seconds) between the rise in [Ca2+] and exocytosis, (2) observation of exocytosis all along the lumen and not clustered around Ca2+ release hot-spots, (3) high affinity (Kd=1.75 microM) Ca2+dependence of exocytosis, (4) significant reduction in exocytosis in the presence of cytosolic EGTA, (5) spatial exclusion of secretory granules from the cell membrane by the endoplasmic reticulum, and (6) inability of local Ca2+ responses to trigger exocytosis. These results strongly indicate that the control of exocytosis, triggered by Ca2+ release from stores, is through the regulation of cytosolic[Ca2+] within a microdomain.

The RCK1 domain of the human BKCa channel transduces Ca2+ binding into structural rearrangements

Large-conductance voltage- and Ca2+-activated K+ (BKCa) channels play a fundamental role in cellular function by integrating information from their voltage and Ca2+ sensors to control membrane potential and Ca2+ homeostasis. The molecular mechanism of Ca2+-dependent regulation of BKCa channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K+ conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BKCa RCK1 domain adopts an α/β fold, binds Ca2+, and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca2+-induced conformational changes in physiologically relevant [Ca2+]. The neutralization of residues known to be involved in high-affinity Ca2+ sensing (D362 and D367) prevented Ca2+-induced structural transitions in RCK1 but did not abolish Ca2+ binding. We provide evidence that the RCK1 domain is a high-affinity Ca2+ sensor that transduces Ca2+ binding into structural rearrangements, likely representing elementary steps in the Ca2+-dependent activation of human BKCa channels.

Molecular Determinants of the CaVβ-induced Plasma Membrane Targeting of the CaV1.2 Channel

Ca(V)beta subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) Ca(V)1 and Ca(V)2 alpha1 subunits. High affinity Ca(V)beta binding onto the so-called alpha interaction domain of the I-II linker of the Ca(V)alpha1 subunit is required for Ca(V)beta modulation of HVA channel gating. It has been suggested, however, that Ca(V)beta-mediated plasma membrane targeting could be uncoupled from Ca(V)beta-mediated modulation of channel gating. In addition to Ca(V)beta, Ca(V)alpha2delta and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca(V)alpha2delta caused a 5-fold stimulation of the whole cell currents measured with Ca(V)1.2 and Ca(V)beta3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca(V)1.2, extracellularly tagged Ca(V)1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca(V)1.2 with either Ca(V)alpha2delta, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca(V)1.2 subunits. In contrast, co-expression with Ca(V)beta3 stimulated plasma membrane expression of Ca(V)1.2 by a 10-fold factor. Mutations within the alpha interaction domain of Ca(V)1.2 or within the nucleotide kinase domain of Ca(V)beta3 disrupted the Ca(V)beta3-induced plasma membrane targeting of Ca(V)1.2. Altogether, these data support a model where high affinity binding of Ca(V)beta to the I-II linker of Ca(V)alpha1 largely accounts for Ca(V)beta-induced plasma membrane targeting of Ca(V)1.2.

The dynamics of mitochondrial Ca 2+ fluxes

We have investigated the kinetics of mitochondrial Ca 2+ influx and efflux and their dependence on cytosolic [Ca 2+] and [Na +] using low-Ca 2+-affinity aequorin. The rate of Ca 2+ release from mitochondria increased linearly with mitochondrial [Ca 2+] ([Ca 2+] M). Na +-dependent Ca 2+ release was predominant al low [Ca 2+] M but saturated at [Ca 2+] M around 400 μM, while Na +-independent Ca 2+ release was very slow at [Ca 2+] M below 200 μM, and then increased at higher [Ca 2+] M, perhaps through the opening of a new pathway. Half-maximal activation of Na +-dependent Ca 2+ release occurred at 5–10 mM [Na +], within the physiological range of cytosolic [Na +]. Ca 2+ entry rates were comparable in size to Ca 2+ exit rates at cytosolic [Ca 2+] ([Ca 2+] c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca 2+] c. As a consequence, the presence of [Na +] considerably reduced the rate of [Ca 2+] M increase at [Ca 2+] c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca 2+] c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca 2+] M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca 2+ buffering, and comparison of our results with data on total mitochondrial Ca 2+ fluxes indicate that the mitochondrial Ca 2+ bound/Ca 2+ free ratio is around 10- to 100-fold for most of the observed [Ca 2+] M range and suggest that massive phosphate precipitation can only occur when [Ca 2+] M reaches the millimolar range.

Identification of a Thiol/Disulfide Redox Switch in the Human BK Channel That Controls Its Affinity for Heme and CO

Heme is a required prosthetic group in many electron transfer proteins and redox enzymes. The human BK channel, which is a large-conductance Ca(2+) and voltage-activated K(+) channel, is involved in the hypoxic response in the carotid body. The BK channel has been shown to bind and undergo inhibition by heme and activation by CO. Furthermore, evidence suggests that human heme oxygenase-2 (HO2) acts as an oxygen sensor and CO donor that can form a protein complex with the BK channel. Here we describe a thiol/disulfide redox switch in the human BK channel and biochemical experiments of heme, CO, and HO2 binding to a 134-residue region within the cytoplasmic domain of the channel. This region, called the heme binding domain (HBD) forms a linker segment between two Ca(2+)-sensing domains (called RCK1 and RCK2) of the BK channel. The HBD includes a CXXCH motif in which histidine serves as the axial heme ligand and the two cysteine residues can form a reversible thiol/disulfide redox switch that regulates affinity of the HBD for heme. The reduced dithiol state binds heme (K(d) = 210 nm) 14-fold more tightly than the oxidized disulfide state. Furthermore, the HBD is shown to tightly bind CO (K(d) = 50 nm) with the Cys residues in the CXXCH motif regulating affinity of the HBD for CO. This HBD is also shown to interact with heme oxygenase-2. We propose that the thiol/disulfide switch in the HBD is a mechanism by which activity of the BK channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions.

Cch1 Restores Intracellular Ca2+ in Fungal Cells during Endoplasmic Reticulum Stress

Pathogens endure and proliferate during infection by exquisitely coping with the many stresses imposed by the host to prevent pathogen survival. Recent evidence has shown that fungal pathogens and yeast respond to insults to the endoplasmic reticulum (ER) by initiating Ca(2+) influx across their plasma membrane. Although the high affinity Ca(2+) channel, Cch1, and its subunit Mid1, have been suggested as the protein complex responsible for mediating Ca(2+) influx, a direct demonstration of the gating mechanism of the Cch1 channel remains elusive. In this first mechanistic study of Cch1 channel activity we show that the Cch1 channel from the model human fungal pathogen, Cryptococcus neoformans, is directly activated by the depletion of intracellular Ca(2+) stores. Electrophysiological analysis revealed that agents that enable ER Ca(2+) store depletion promote the development of whole cell inward Ca(2+) currents through Cch1 that are effectively blocked by La(3+) and dependent on the presence of Mid1. Cch1 is permeable to both Ca(2+) and Ba(2+); however, unexpectedly, in contrast to Ca(2+) currents, Ba(2+) currents are steeply voltage-dependent. Cch1 maintains a strong Ca(2+) selectivity even in the presence of high concentrations of monovalent ions. Single channel analysis indicated that Cch1 channel conductance is small, similar to that reported for the Ca(2+) current I(CRAC). This study demonstrates that Cch1 functions as a store-operated Ca(2+)-selective channel that is gated by intracellular Ca(2+) depletion. The inability of cryptococcal cells that lacked the Cch1-Mid1 channel to survive ER stress suggests that Cch1 and its co-regulator, Mid1, are critical players in the restoration of Ca(2+) homeostasis.

An encounter of the third kind without the usual suspects: coherent activation of asynchronous transmitter release independent of presynaptic excitation

An encounter of the third kind without the usual suspects: coherent activation of asynchronous transmitter release independent of presynaptic excitation

Doc2b is a high-affinity Ca2+ sensor for spontaneous neurotransmitter release.

Synaptic vesicle fusion in brain synapses occurs in phases that are either tightly coupled to action potentials (synchronous), immediately following action potentials (asynchronous), or as stochastic events in the absence of action potentials (spontaneous). Synaptotagmin-1, -2, and -9 are vesicle-associated Ca2+ sensors for synchronous release. Here we found that double C2 domain (Doc2) proteins act as Ca2+ sensors to trigger spontaneous release. Although Doc2 proteins are cytosolic, they function analogously to synaptotagmin-1 but with a higher Ca2+ sensitivity. Doc2 proteins bound to N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complexes in competition with synaptotagmin-1. Thus, different classes of multiple C2 domain-containing molecules trigger synchronous versus spontaneous fusion, which suggests a general mechanism for synaptic vesicle fusion triggered by the combined actions of SNAREs and multiple C2 domain-containing proteins.

Plasma membrane Ca2+-ATPases in the nervous system during development and ageing

Calcium signaling is used by neurons to control a variety of functions, including cellular differentiation, synaptic maturation, neurotransmitter release, intracellular signaling and cell death. This review focuses on one of the most important Ca(2+) regulators in the cell, the plasma membrane Ca(2+)-ATPase (PMCA), which has a high affinity for Ca(2+) and is widely expressed in brain. The ontogeny of PMCA isoforms, linked to specific requirements of Ca(2+) during development of different brain areas, is addressed, as well as their function in the adult tissue. This is based on the high diversity of variants in the PMCA family in brain, which show particular kinetic differences possibly related to specific localizations and functions of the cell. Conversely, alterations in the activity of PMCAs could lead to changes in Ca(2+) homeostasis and, consequently, to neural dysfunction. The involvement of PMCA isoforms in certain neuropathologies and in brain ageing is also discussed.

Voltage Sensor Activation Facilitates Magnesium-Gated Channel Opening in BK Channels

Under physiological conditions, Mg2+ is an intracellular activator of Ca2+- and voltage-activated potassium (BK) channels. To investigate gating by Mg2+ acting through its low affinity site located under each S4 voltage sensor (Yang et al. 2007, 2008; Horrigan & Ma 2008), we studied a BK channel in which the high affinity Ca2+ sites in both the RCK1 domain and the calcium bowl were disabled by mutation. Using single-channel recording from inside-out patches to measure channel activation after voltage steps from −100 mV to +100 mV, we found that 10 mM Mg2+ reduces the latency to first opening after the voltage step and increases channel activation through an increase in the number of openings per burst and mean open duration. This suggests that Mg2+ can bind to both closed and open states of the channel, but it is not clear whether the closed-state binding occurs at the negative or positive potential. Therefore we recorded single-channel activity in macro-patches held at a constant −50 or −100 mV. At −50 mV, when the voltage sensors are occasionally activated, 10 mM Mg2+ decreases the duration of the closed intervals between bursts of activity and increases burst duration through an increase in the number of openings per burst and mean open duration. At −100 mV, when the voltages sensors are mainly deactivated, 10 mM Mg2+ has little effect on the closed intervals between bursts or mean open duration, and most of the bursts are unitary, consisting of a single brief opening. The above data are consistent with a model in which Mg2+ can bind to the BK channel in the closed conformation when the voltage sensors are activated. The bound Mg2+ then facilitates opening. Supported by NIH grant AR32805.

Impairment of PMCA Activity by Amyloid β-Peptide in Membranes from Alzheimer's Disease-Affected Brain and from Other Model Systems

High-affinity Ca2+-transport ATPases are key components in the machinery of cytosolic Ca2+ regulation. We have found that the Ca2+-dependence of plasma membrane Ca2+-ATPase (PMCA) activity is constant between pCa values of 6.5 and 4.5 in membranes from Alzheimer's disease (AD) affected-brain whereas in normal brain the activity increases from pCa 6.5 to 5.5 and then decreases from 5.5 to 4.5. However, the Ca2+-dependencies of sarco(endo)plasmic- (SERCA) and secretory pathway- (SPCA) Ca2+-ATPases activity are similar in AD and normal brains.

Cch1 Restores Intracellular Calcium in Fungal Cells during ER Stress

Pathogens endure and proliferate during infection by exquisitely coping with the many stresses imposed by the host as a means to prevent pathogen survival. Recent evidence has shown that fungal pathogens and yeast respond to insults to the ER (endoplasmic reticulum) by initiating Ca2+ influx across their plasma membrane. Although the high-affinity Ca2+ channel, Cch1 and its subunit Mid1, have been suggested as the protein complex responsible for mediating Ca2+ influx, a direct demonstration of the gating mechanism of the Cch1 channel remains elusive. In this first mechanistic study of Cch1 channel activity we show that the Cch1 channel from the model human fungal pathogen, Cryptococcus neoformans, is directly activated by the depletion of intracellular Ca2+ stores. Electrophysiological analysis revealed that agents that enable ER Ca2+ store depletion promote the development of whole-cell inward Ca2+ currents through Cch1 that are effectively blocked by La3+ and dependent on the presence of Mid1. Cch1 is permeable to both Ca2+ and Ba2+ however, unexpectedly, in contrast to Ca2+ currents, Ba2+ currents are steeply voltage-dependent. Cch1 maintains a strong Ca2+ selectivity even in the presence of high concentrations of monovalent ions. Single channel analysis indicated that Cch1 channel conductance is small, similar to that reported for the Ca2+ current ICRAC. This study demonstrates that Cch1 functions as a store-operated Ca2+-selective channel that is gated by intracellular Ca2+ depletion. In ER stress conditions, Cch1 is poised to restore Ca2+ homeostasis and consequently fungal pathogens like C. neoformans require Cch1 activity for survival and colonization of the host.

Conformational Changes in Guanylate Cyclase-Activating Protein 1 Induced by Ca2+ and N-Terminal Fatty Acid Acylation

Neuronal Ca(2+) sensors (NCS) are high-affinity Ca(2+)-binding proteins critical for regulating a vast range of physiological processes. Guanylate cyclase-activating proteins (GCAPs) are members of the NCS family responsible for activating retinal guanylate cyclases (GCs) at low Ca(2+) concentrations, triggering synthesis of cGMP and recovery of photoreceptor cells to the dark-adapted state. Here we use amide hydrogen-deuterium exchange and radiolytic labeling, and molecular dynamics simulations to study conformational changes induced by Ca(2+) and modulated by the N-terminal myristoyl group. Our data on the conformational dynamics of GCAP1 in solution suggest that Ca(2+) stabilizes the protein but induces relatively small changes in the domain structure; however, loss of Ca(+2) mediates a significant global relaxation and movement of N- and C-terminal domains. This model and the previously described "calcium-myristoyl switch" proposed for recoverin indicate significant diversity in conformational changes among these highly homologous NCS proteins with distinct functions.

Two Photon Imaging of Calcium Signalling at the Mouse Inner Hair Cell Ribbon Synapse

The information sent from each cochlear inner hair cell (IHC) to the afferent nerve is determined by 10-20 ribbon synapses, structures specialised for rapid release of vesicles upon cell depolarization. To study the IHC calcium domains during transmitter release in mature wild-type mice, we have imaged and simultaneously measured currents in IHCs through an apical opening in the isolated temporal bone. Cells were recorded on the stage of an upright 2PCLSM at room temperature, superfused with medium containing 2mM Ca2+. IHCs could be visualised either with oblique optics or by using 830nm trans-illumination through bone structures. Using whole-cell tight seal recording, with Cs+ containing pipettes to reduce large outward currents, the I-V curve of the IHCs exhibit a Ca current with peak magnitude of approx 80pA near −20mV. To observe the distribution of Ca2+ entry in the vicinity of the ribbon sites, cells we pipette-loaded IHCs with either high or low affinity Ca2+ dyes (200uM OGB1 or OGB5N respectively) and imaged the basal IHC pole up to maximal rates of 70 frames/s during 20 ms or 100ms depolarizing steps to 0mV. At the fastest rates, the images derived from within single cells showed an initial punctuate rise of Ca2+ at the presumed synaptic sites with a larger increase at the neural side, a possible correlate of differing afferent thresholds known to characterise auditory nerve fibres. The sites were correlated with fluorescent hotspot distribution identified by IHC FM-dye uptake. The distribution of sites, the localisation of signal maxima close to (<3um) the plasma-membrane and recovery time constant (∼100ms) of Ca2+ influx also suggests that intrinsic Ca2+ buffering near the ribbon synapse was not significantly perturbed. Supported by EuroHear, the Physiological Society (SC), and College de France (JBM).

Novel Insights into the Role of Junctate in Calcium Homeostasis: Identification of Binding Domain on the InsP3R and Cellular Localization as Determined by TIRF Microscopy

Junctate is a 33 kDa calcium binding protein with a single ER/SR membrane spanning domain expressed in excitable and non-excitable tissues. It is generated by alternative splicing from the AsH-J-J locus, which also encodes the enzyme aspartyl-s-hydroxylase, the sarcoplasimc reticulum structural protein junctin and humbug, a truncated version of aspartyl-s-hydroxylase, lacking its catalytic domain, which shares with junctate the high capacity moderate affinity Ca2+ binding domain and is over-expressed in a variety of tumours. We have previously shown that junctate forms a macromolecular complex with the InsP3R and TRPC3 channels and when transiently over-expressed in HEK293 cells, it induces extensive proliferation of the ER resulting in significantly larger and more frequent couplings between the ER and the plasma membrane. In the present work we have mapped the binding domain of the cytoplasmic NH2 terminus of junctate on the InsP3R and show that it binds to the domain involved in InsP3 binding. Such a result is supported by the finding that in the presence of a peptide encompassing the NH2 terminal domain of junctate, the Bmax for InsP3 binding is significantly higher than that obtained in the presence of an unrelated peptide. Transmission electron microscopy revealed that clones stably transfected with junctate-YFP display a significant larger number of junctions between the ER and the plasma membrane compared to control HEK293 cells and this effect is enhanced in clones that also over-express TRCP3. The size and distribution of these punctae however, was not affected by the addition of agonists mobilizing calcium via InsP3R activation.

Ca2+-Induced Conformational Changes of Na+-Ca2+ Exchanger Dimers: Role of Ca2+ Binding Domains

The Na+-Ca2+ exchanger is activated by the binding of cytoplasmic Ca2+ to two Ca2+ binding domains (CBD1 and CBD2). How binding of Ca2+ is translated into exchanger activation is unknown. We investigated Ca2+-dependent movements as changes in FRET between exchanger dimers tagged with CFP or YFP at positions 266 within the large cytoplasmic loop of NCX1.1. The biophysical properties of the fluorescent exchangers are identical to those of the untagged NCX. Fluorescent exchangers were coexpressed in Xenopus oocytes from which plasma membrane sheets were isolated. Upon addition of Ca2+, the coexpressed pair NCX-266CFP + NCX-266YFP showed an increase in FRET in a dose-dependent manner. Similar FRET changes were observed after mutating the Ca2+ coordination site in CBD2 (E516L). Exchanger E516L is not Ca2+ regulated. In contrast, mutating the Ca2+ coordination site in CBD1 (D421A, E451A and D500V) abolished FRET changes. These residues likely disrupt binding of Ca2+ to CBD1. Nevertheless, Ca2+ regulation of NCX is retained though with a substantial decrease in apparent affinity for Ca2+. These results indicate that the Ca2+-induced conformational changes of NCX dimers arise exclusively from the movement of CBD1. Peptides of Ca2+ binding domains, flanked by CFP and YFP, recapitulated the full length exchanger results: CBD1 showed movement upon Ca2+ addition while CBD2 did not. A peptide spanning CBD1-CBD2 displayed Ca2+-dependent movement, which was abolished by mutating the Ca2+ coordination site in CBD1. Our results indicate the following: 1. Exchanger conformational changes are associated with the occupancy of a high affinity Ca2+ binding site exclusively within CBD1. 2. FRET studies confirm that the Na+-Ca2+ exchanger exists as a dimer.

Mechanisms of Spontaneous Calcium Wave Generation During Beta-Adrenergic Stimulation in Rabbit Ventricular Myocytes

The beta-adrenergic signaling pathway represents the principal positive inotropic mechanism of the heart. While the effects of beta-adrenergic stimulation on L-type Ca channel Ca influx and SERCA-mediated sarcoplasmic reticulum (SR) Ca uptake are well established, the effects on SR Ca release through ryanodine receptor (RyR) release clusters remains highly controversial. Here, we examine SR Ca release in rabbit ventricular myocytes in the form of spontaneous Ca waves during beta-adrenergic stimulation with isoproterenol under controlled cytosolic and SR [Ca]. Cytosolic Ca was monitored using high-affinity Ca indicators indo-1 or rhod-2, while SR Ca was measured directly using the low-affinity Ca indicator fluo-5N or indirectly using the amplitude of the cytosolic Ca transient in response to 10 mM caffeine. Under control conditions, Ca waves were not observed following rest from 0.75 Hz pacing. In the presence of isoproterenol (500 nM), SR Ca content increased by 34% and spontaneous Ca waves were observed in 67% of cells during rest after pacing. However, when post-rest cytosolic Ca and SR Ca content were experimentally matched to control conditions using low extracellular Ca (100 uM versus 2 mM) and SERCA inhibition (7.5 uM cyclopiazonic acid), spontaneous Ca waves were never observed in the presence of isoproterenol. In contrast, pharmacological sensitization of the RyR with 250 uM caffeine induced Ca waves under control conditions (8/12 cells) and in the presence of isoproterenol at matched cytosolic Ca and SR Ca content (7/12). Together, these data suggest that spontaneous Ca release during beta-adrenergic stimulation is a result of increased RyR sensitivity in response to increased SR Ca content, and is not due to direct alterations in RyR function by the beta-adrenergic signaling cascade.