High-affinity Ca2 Research Articles

Sevoflurane modulation of Ca2+ regulation in skeletal muscle sarcoplasmic reticulum vesicles from young and mature rabbits

Developmental differences in splice variants of the two key sarcoplasmic reticulum (SR) calcium regulatory proteins, ryanodine (RyR1), and sarcoendoplasmic reticulum calcium pump (SERCA1) have been linked to various neuromuscular disorders, but not malignant hyperthermia (MH). However, it is unclear whether an age-related difference in volatile anesthetic-mediated SR calcium function exists that could add to our current understanding of the clinical presentation of MH syndrome and provide insight into molecular mechanisms for general anesthesia that may have other physiologic and/or pathophysiologic significance. Therefore, the effects of sevoflurane on intracellular calcium regulation in isolated SR membrane vesicles from the skeletal muscle of healthy young rabbits were compared to their adult counterpart using an established in vitro model with the assumption that exposure to sevoflurane would elicit a weaker response in the young SR. Through dual wavelength spectroscopy of Ca(2+): Arsenazo III difference absorbance, the effects of sevoflurane on SR Ca(2+) uptake rate and release in heavy and light fraction SR membrane vesicles isolated from the white muscle of anesthetized, postweaned (age = 6 weeks, n = 5) and adult (age = 6 months, n = 5) male New Zealand rabbits were examined. The adult group showed a 50% increase in Ca(2+) uptake rate from control at both subclinical and clinically relevant anesthetic concentrations, whereas in the SR from the younger animals, Ca(2+) uptake rate was not altered by any concentration of sevoflurane. The sensitivity of both the low and high affinity Ca(2+)-binding sites on RyR1 was increased by sevoflurane to the same extent in the SR vesicles from the young and mature adult rabbits. Interestingly, a greater potency of sevoflurane for the high affinity-binding site was identified, and this was independent of age. These findings suggest that the sensitivity of the SR to sevoflurane-mediated Ca(2+) uptake may be increased with maturity, while an analogous developmental effect on RyR1 is less probable. Nonetheless, this study shows for the first time that a potent inhalational agent such as sevoflurane can influence the high affinity SR calcium-binding site by lowering the extraluminal concentration of calcium necessary to trigger calcium release. While this may not be of consequence when inhaled anesthetics are administered to normal children or adults, it may have life-threatening consequences in carriers of RyR1 mutations.

Calcium Pumps in Health and Disease

Ca2+-ATPases (pumps) are key actors in the regulation of Ca2+ in eukaryotic cells and are thus essential to the correct functioning of the cell machinery. They have high affinity for Ca2+ and can efficiently regulate it down to very low concentration levels. Two of the pumps have been known for decades (the SERCA and PMCA pumps); one (the SPCA pump) has only become known recently. Each pump is the product of a multigene family, the number of isoforms being further increased by alternative splicing of the primary transcripts. The three pumps share the basic features of the catalytic mechanism but differ in a number of properties related to tissue distribution, regulation, and role in the cellular homeostasis of Ca2+. The molecular understanding of the function of the pumps has received great impetus from the solution of the three-dimensional structure of one of them, the SERCA pump. These spectacular advances in the structure and molecular mechanism of the pumps have been accompanied by the emergence and rapid expansion of the topic of pump malfunction, which has paralleled the rapid expansion of knowledge in the topic of Ca2+-signaling dysfunction. Most of the pump defects described so far are genetic: when they are very severe, they produce gross and global disturbances of Ca2+ homeostasis that are incompatible with cell life. However, pump defects may also be of a type that produce subtler, often tissue-specific disturbances that affect individual components of the Ca2+-controlling and/or processing machinery. They do not bring cells to immediate death but seriously compromise their normal functioning.

Ontogeny of ATP hydrolysis and isoform expression of the Plasma Membrane Ca2+-ATPase in mouse brain

BackgroundPlasma membrane Ca2+-ATPases (PMCAs) are high affinity Ca2+ transporters actively involved in intracellular Ca2+ homeostasis. Considering the critical role of Ca2+ signalling in neuronal development and plasticity, we have analyzed PMCA-mediated Ca2+-ATPase activity and PMCA-isoform content in membranes from mouse cortex, hippocampus and cerebellum during postnatal development.ResultsPMCA activity was detected from birth, with a faster evolution in cortex than in hippocampus and cerebellum. Western blots revealed the presence of the four isoforms in all regions, with similar increase in their expression patterns as those seen for the activity profile. Immunohistochemistry assays in cortex and hippocampus showed co-expression of all isoforms in the neuropil associated with synapses and in the plasma membrane of pyramidal cells soma, while cerebellum showed a more isoform-specific distribution pattern in Purkinje cells.ConclusionThese results show an upregulation of PMCA activity and PMCA isoforms expression during brain development in mouse, with specific localizations mainly in cerebellum. Overall, our findings support a close relationship between the ontogeny of PMCA isoforms and specific requirements of Ca2+ during development of different brain areas.

Functional Roles of EF-Hands in Human Potassium Channel-Interacting Protein 2.2

Single site-directed mutations at each of the four EF-hand loops of potassium channel-interacting protein 2.2 (KChIP2.2) were carried out to explore the functional roles of EF-hands in KChIP2.2. In contrast to those on EF-hands 1 and 2, mutations on EF-hands 3 or 4 distorted the high affinity Ca(2+)-binding site of KChIP2.2. However, the Mg(2+)-binding ability of KChIP2.2 was marginally affected by the mutations. The gross conformation of mutated KChIP2.2 was indistinguishable from wild-type KChIP2.2 as revealed by CD spectra. The results of size exclusion chromatography showed that, with exception of EF-hand 4 mutant, mutations on EF-hands 1, 2 or 3 caused KChIP2.2 to form oligomer. Pull-down assay revealed that, unlike wild-type KChIP2.2, the interaction between mutated KChIP2.2 and Kv4.2 was not notably enhanced by Ca(2+) and Mg(2+). Coexpression of Kv4.2 and KChIP2.2 in HeLa cells revealed that mutations on EF-hands did not alter the intracellular co-localization of KChIP2.2 and Kv4.2. Together with previous findings that EF-hand mutants of KChIP proteins are unable to regulate the kinetics of Kv4.2, our data show that the intact EF-hands should be crucial for the formation of active conformation of KChIP2.2 when the protein is loaded with Ca(2+) and Mg(2+).

Otoferlin is critical for a highly sensitive and linear calcium-dependent exocytosis at vestibular hair cell ribbon synapses.

Otoferlin, a C2-domain-containing Ca(2+) binding protein, is required for synaptic exocytosis in auditory hair cells. However, its exact role remains essentially unknown. Intriguingly enough, no balance defect has been observed in otoferlin-deficient (Otof(-/-)) mice. Here, we show that the vestibular nerve compound action potentials evoked during transient linear acceleration ramps in Otof(-/-) mice display higher threshold, lower amplitude, and increased latency compared with wild-type mice. Using patch-clamp capacitance measurement in intact utricles, we show that type I and type II hair cells display a remarkable linear transfer function between Ca(2+) entry, flowing through voltage-activated Ca(2+) channels, and exocytosis. This linear Ca(2+) dependence was observed when changing the Ca(2+) channel open probability or the Ca(2+) flux per channel during various test potentials. In Otof(-/-) hair cells, exocytosis displays slower kinetics, reduced Ca(2+) sensitivity, and nonlinear Ca(2+) dependence, despite morphologically normal synapses and normal Ca(2+) currents. We conclude that otoferlin is essential for a high-affinity Ca(2+) sensor function that allows efficient and linear encoding of low-intensity stimuli at the vestibular hair cell synapse.

Mechanisms of after‐hyperpolarization following activation of fly visual motion‐sensitive neurons

In many neurons, strong excitatory stimulation causes an after-hyperpolarization (AHP) at stimulus offset, which might give rise to activity-dependent adaptation. Graded-potential visual motion-sensitive neurons of the fly Calliphora vicina respond with depolarization and hyperpolarization during motion in their preferred direction and their anti-preferred direction, respectively. A prominent after-response, opposite in sign to the response during motion, is selectively expressed after stimulation with preferred-direction motion. Previous findings suggested that this AHP is generated in the motion-sensitive neurons themselves rather than in presynaptic processing layers. However, it remained unknown whether the AHP is caused by membrane depolarization itself or by another process, e.g. a signaling cascade triggered by activity of excitatory input channels. Here we showed by current injections and voltage clamp that the AHP and a corresponding current are generated directly by depolarization. To test whether the generation of an AHP is linked to depolarization via a Ca(2+)-dependent mechanism, we used photoactivation of a high-affinity Ca(2+) buffer. In accordance with previous findings the AHP was insensitive to manipulation of cytosolic Ca(2+). We propose that membrane depolarization presents a more direction-selective mechanism for the control of AHP than other potential control parameters.

Comparative effects of H+ and Ca2+ on large-conductance Ca2+- and voltage-gated Slo1 K+ channels

Large-conductance Ca2+- and voltage-gated Slo1 BK channels are allosterically activated by depolarization and intracellular ligands such as Ca2+. Of the two high-affinity Ca2+ sensors present in the channel, the RCK1 sensor also mediates H+-dependent activation of the channel. In this study, we examined the comparative mechanisms of the channel activation by Ca2+ and H+. Steady-state macroscopic conductance-voltage measurements as well as single-channel openings at negative voltages where voltage-sensor activation is negligible showed that at respective saturating concentrations Ca2+ is more effective in relative stabilization of the open conformation than H+. Calculations using the Debye-Hückel formulation suggest that small structural changes in the RCK1 sensor, on the order of few angstroms, may accompany the H+-mediated opening of the channel. While the efficacy of H+ in activation of the channel is less than that of Ca2+, H+ more effectively accelerates the activation kinetics when examined at the concentrations equipotent on macroscopic voltage-dependent activation. The RCK1 sensor therefore is capable of transducing the nature of the ligand bound and transmits qualitatively different information to the channel's permeation gate.

The functional expression of toxic genes: Lessons learned from molecular cloning of CCH1, a high-affinity Ca 2+ channel

Some genes cannot be cloned by conventional methods because in most cases the genes or gene products are toxic to Escherichia coli. CCH1 is a high-affinity Ca 2+ channel present in the plasma membrane of Cryptococcus neoformans and other fungi. Like many toxic genes, the molecular cloning of CCH1 has been a major challenge; consequently, direct studies of CCH1 channel activity in heterologous expression systems have been impossible. We have devised a straightforward approach that resulted in the molecular cloning and functional expression of CCH1 by exploiting homologous recombination both in vitro and in vivo. This approach precluded the standard enzyme digestion-mediated ligation reactions and the subsequent isolation of plasmids from E. coli. The shuttle plasmid carrying CCH1–GFP, which was prepared in vitro and propagated in yeast, was successfully expressed in the mammalian cell line HEK293 (human embryonic kidney 293). CCH1 transcripts were detected only in HEK293 cells transfected with the plasmid DNA. Fluorescence microscopy studies revealed the expression of CCH1–GFP fusion protein on the cell surface of HEK293 cells, similar to the localization pattern of a well-characterized plasma membrane-associated K + channel. This approach will be particularly useful for genes that encode ion channels and transporters that cannot be cloned by conventional techniques requiring E. coli.

A Functional and Structural Study of Troponin C Mutations Related to Hypertrophic Cardiomyopathy

Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca(2+) sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281-288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca(2+)] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca(2+) sensitivity with only A8V demonstrating lowered Ca(2+) sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca(2+). None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca(2+) affinity of site II measured by 2-(4'-(2''-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca(2+). Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca(2+) sensitivity of the myofilament; 2) the effects of the mutations on the Ca(2+) affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca(2+) sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca(2+) binding to cTnC.

Synaptotagmin-2 Controls Regulated Exocytosis but Not Other Secretory Responses of Mast Cells

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

Kinetic and Equilibrium Properties of Regulatory Calcium Sensors of NCX1 Protein

The crystal structures of the CBD1 and CBD2 domains of the Na+/Ca2+ exchanger protein (NCX1) provided a major breakthrough in Ca2+-dependent regulation of NCX1, although the dynamic aspects of the underlying molecular mechanisms are still not clear. Here we provide new experimental approaches for evaluating the kinetic and equilibrium properties of Ca2+ interaction with regulatory sites by using purified preparations of CBD1, CBD2, and CBD12 proteins. CBD12 binds approximately 6 Ca2+ ions (mol/mol), whereas the binding of only approximately 2 Ca2+ ions is observed (with a Hill coefficient of nH=approximately 2) either for CBD1 or CBD2. In the absence of Mg2+, CBD1 has a much higher affinity for Ca2+ (Kd=0.3+/-1.2 microm) than CBD2 (Kd=5.0+/-1.2 microm). The Ca2+ dissociation from CBD2 (koff=230+/-70 s(-1)) is at least 25 times faster than from CBD1 (koff=10+/-3 s(-1)), whereas the kon values indicate fast kinetics for Ca2+ binding (kon=koff/Kd=10(7)-10(8) m(-1) s(-1)) for both CBDs. At 2-5 mm Mg2+, both CBDs bind Ca2+, with a Kd of 1-2 microm (Mg2+ has very little effect on Ca2+ off rates). Mg2+ cannot occupy the primary site of CBD2, whereas the other Ca2+ sites of CBDs interact with Mg2+ as well. There is no competition between Na+ and Ca2+ for any CBD site. The kinetically diverse Ca2+ sensors may sense differentially the dynamic swings in [Ca2+] within specific subcellular compartments (dyadic cleft, submembrane space, bulk cytosol, etc.).

The mechanisms of multi-component paired-pulse facilitation of neurotransmitter release at the frog neuromuscular junction

We have studied the mechanisms of paired-pulse facilitation (PPF) of neurotransmitter release in isolated nerve-muscle preparations of the frog cutaneous pectoris muscle. In normal extracellular Ca(2+) concentration ([Ca(2+)](o), 1.8 mM), as the interpulse interval was increased from 5 to 500 ms, PPF decayed as a sum of two exponential components: a larger but shorter first component (F1) and a smaller but more prolonged second component (F2). In low [Ca(2+)](o) (0.5 mM), both F1 and F2 increased, and a third "early" component (Fe) appeared whose amplitude was larger and whose duration was shorter than F1 or F2. In the presence of the "fast" Ca(2+) buffer BAPTA-AM, Fe disappeared, whereas F1 and F2 decreased in amplitude and duration. In contrast, the "slow" Ca(2+) buffer EGTA-AM caused a decrease of Fe and reduction or complete blockade of F2, without any changes of F1. In solutions containing Sr(2+) (1 mM), the magnitude of Fe was decreased, F1 was significantly reduced and shortened, but F2 was unaffected. Application of the calmodulin inhibitor W-7 (10 microM) at normal [Ca(2+)](o) produced a marked decrease of F2, and at low [Ca(2+)](o), a complete blockade of Fe. These results suggest that PPF at frog motor nerve terminals is mediated by several specific for different PPF components intraterminal Ca(2+) binding sites, which trigger neurotransmitter release. These sites have a higher affinity for Ca(2+) ions and are located farther from the release-controlling Ca(2+) channels than the Ca (2+) sensor that mediates phasic release.

Highlights From The Literature

Highlights From The Literature

Calcium Sensing Properties of the RCK1 Domain of the Human BK Channel: Effects of the D362/367A Mutation

Recent studies suggest that Ca2+-dependent activation of the large-conductance voltage- and Ca2+-activated K+ (BK) channel is controlled by two RCK (Regulators of K+ Conductance) domains located in the C-terminus, encompassing high affinity Ca2+-sites D362/367 and M513 in RCK1, and a Ca2+-bowl (D894-898) in RCK2 (Schreiber, et al, 1999, Lingle et al. 2002, Bao et al. 2004). Previously, we characterized Ca2+-induced conformational changes and the role of the Ca2+ bowl in the hSloRCK2 domain using solution-based analysis (Yusifov et al., PNAS 2008).Using a similar approach, we are now investigating the Ca2+-dependent properties of the WT and D362/367A mutations of a purified protein corresponding to the amino acid sequence (322IIE¼DPL667) that forms the human BK channel's RCK1 domain.The calcium-binding activity of purified RCK1 (10ug), loaded on a nitrocellulose membrane, was directly probed by dot blot analysis of 45Ca2+-binding. Albumin and Troponin were used as negative and positive controls, respectively. RCK1 showed remarkable calcium-binding ability when compared to Albumin.Circular Dichroism (CD) analysis of the WT-RCK1 revealed a calcium-dependent spectral change, corresponding to an increased β-strand content of ∼9% as the free [Ca2+] was increased from 0.015 to 31.2μM. This change was paralleled by a similar decrease in α-helix content, while the turns and unordered fractions remained practically unchanged.On the other hand, the CD spectra of RCK1-D362/367A mutant displays ∼7-8% increased beta content, similar to the calcium-bound form of WT-RCK1. The Far-UV CD spectra obtained of hSloRCK1-D362/367A mutant in increasing free Ca2+ displayed no changes, suggesting a lack of substantial Ca2+-dependent structural changes. Based on these findings, we propose that the D362/367A mutation in hSloRCK1 may lead to a conformational state of hSloRCK1 that is unable to translate Ca2+-binding to channel gating.

Comparative Mechanisms Of Activation Of The Slo1 BK Channel By Ca2+ And H+ Mediated By The RCK1 Domain

Large-conductance Ca2± and voltage-gated K+ (Slo1 BK) channels are allosterically activated by depolarization and intracellular Ca2+. High-affinity activation of the channel by Ca2+ involves two sites, the Ca2+ bowl sensor and the RCK1 sensor, the latter of which is also required for the stimulatory action of intracellular H+ (Hou et al., Nat Struct Mol Biol. 15, 403, 2008). We investigated the comparative effects of Ca2+ and H+ on activation of the Slo1 BK channel mediated by the RCK1 sensor using a Ca2+ bowl-defective mutant expressed in HEK cells. Decreasing pHi from 7.5 to 6.2 shifted the voltage-conductance (GV) curve to the left by ∼50 mV. The shift in GV by H+ was, however, only ∼40% of that caused by a saturating concentration of Ca2+ in the mutant. Single-channel measurements at negative voltages where voltage sensor activation is negligible verified that 200 uM Ca2+ drastically increased open probability, corresponding to the allosteric coupling factor C = ∼4 in the model of Horrigan and Aldrich (J Gen Physiol. 120, 267, 2002). In contrast, at such negative voltages, H+ at pH = 6.2 was much less effective in increasing open probability, leading to the estimated C value = ∼1.3, placing H+ between Ca2+ and Mg2+ in the ability to promote opening in the absence of voltage sensor activation. Likewise, at the respective saturation concentrations, H+ was less effective than Ca2+ at regulating channel kinetics. Our electrophysiological measurements and simulations collectively suggest that H+ is a partial agonist of the RCK1 high-affinity Ca2+ sensor and that a small change in the relative position of His and Asp residues in the sensor, ∼0.1 nm, may underlie the activation of the channel by H+. Supported by NIH.

Which Low-affinity Fluorescent Calcium Indicators Accurately Track The Change In Myoplasmic Free Calcium Concentration (Δ[Ca]) In Skeletal Muscle?

In vertebrate twitch fibers, spatially-averaged Δ[Ca] elicited by an action potential (AP) is large and brief. Consequently, Δ[Ca] is more accurately measured with low-affinity than with high-affinity Ca indicators (Hirota et al., 1989; Baylor and Hollingworth, 1998). Previous studies with low-affinity fluorescent indicators found that the time course of Δ[Ca] is quite accurately monitored with furaptra (= mag-fura-2), mag-fura-5, and mag-indo-1 (Konishi et al., 1991; Zhao et al, 1996). Because these tri-carboxylate Ca indicators have some sensitivity to free magnesium ([Mg]), we have evaluated three low-affinity tetra-carboxylate indicators, fura-5N, OGB-5N, and fluo-5N, which have negligible [Mg] sensitivity. To do so, resting fluorescence (FR) and fluorescence changes elicited by an AP (ΔF) were measured at 16 °C in frog single fibers micro-injected with both furaptra and one of the other three indicators. Disappointingly, with the other three indicators, the full-duration at half maximum of ΔF was larger than that with furaptra, on average, by 37, 51, and 53%, respectively, increases that do not appear to arise from increased saturation of indicator with Ca. We also evaluated mag-fluo-4 (cf. Caputo et al., 2004), another tri-carboxylate indicator. Encouragingly, mag-fluo-4's ΔF time course was essentially identical to furaptra's while its signal-noise ratio with visible excitation wavelengths was an order of magnitude larger (for similar concentrations of indicator). However, because FR of mag-fluo-4 probably arises largely from indicator molecules bound with Mg, calibration of mag-fluo-4's ΔF/FR in terms of Δ[Ca] is likely to be more sensitive to variations in [Mg]. Also, with mag-fluo-4, unlike furaptra, fluorescence anisotropy values varied with the plane of polarization of the exciting light, thus revealing a population of mag-fluo-4 molecules that are oriented and presumably bound to structural components within the fiber.

Fast-fluorescence dynamics in nonratiometric calcium indicators

The fluorescence decay of high-affinity nonratiometric Ca2+ indicator Oregon Green BAPTA-1 (OGB-1) is analyzed with unprecedented temporal resolution in the two-photon excitation regime. A triple exponential decay is shown to best fit the fluorescence dynamics of OGB-1. We provide a model for accurate measurements of the free Ca2+ concentration and dissociation constants of nonratiometric calcium indicators.

A functional calcium-binding site in the metalloprotease domain of ADAMTS13

AbstractADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca2+ ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca2+ concentrations, a high-affinity functional ADAMTS13 Ca2+-binding site was suggested with KD(app) of 80 μM (± 15 μM) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca2+-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca2+ dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca2+-binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the KD(app) for Ca2+ of ADAMTS13, and for D187A and E212A also reduced the Vmax to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca2+ revealed an approximately 13-fold reduction in specificity constant, kcat/Km, contributed by changes in both Km and kcat. These results were corroborated using plasma-purified VWF as a substrate. Together, our results demonstrate that a major influence of Ca2+ upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft.

Altered Ca 2+ dependence of synaptosomal plasma membrane Ca 2+ ‐ATPase in human brain affected by Alzheimer's disease

High-affinity Ca(2+) transport ATPases play a crucial role in controlling cytosolic Ca(2+). The amyloid beta-peptide (Abeta) is a neurotoxic agent found in affected neurons in Alzheimer's disease (AD) that has been implicated in dysregulation of Ca(2+) homeostasis. Using kinetic assays, we have shown that the Ca(2+) dependencies of intracellular Ca(2+)-ATPase (SERCA and SPCA) activity are the same in human AD and normal brain but that of plasma membrane Ca(2+)-ATPase (PMCA) is different. The addition of Abeta to normal brain decreases the PMCA activity measured at pCa 5.5, resulting in the same Ca(2+)dependency as that seen in AD brain, whereas the addition of Abeta to AD brain has no effect on PMCA activity. Abeta also decreases the activity of PMCA purified from pig cerebrum, the effect being isoform specific. The level of inhibition of purified PMCA caused by Abeta is reduced by cholesterol, and the level of inhibition of PMCA activity by Abeta in the raft fraction of pig synaptosomal membranes is lower than for the nonraft fraction. We conclude that the effect of Abeta on PMCA activity could be important in amyloid toxicity, resulting in cytoplasmic Ca(2+) dysregulation and could explain the different Ca(2+) dependencies of PMCA activity observed in normal and AD brain.

Measuring the Influence of the BKCa β1 Subunit on Ca2+ Binding to the BKCa Channel

The large-conductance Ca2+-activated potassium (BKCa) channel of smooth muscle is unusually sensitive to Ca2+ as compared with the BKCa channels of brain and skeletal muscle. This is due to the tissue-specific expression of the BKCa auxiliary subunit β1, whose presence dramatically increases both the potency and efficacy of Ca2+ in promoting channel opening. β1 contains no Ca2+ binding sites of its own, and thus the mechanism by which it increases the BKCa channel's Ca2+ sensitivity has been of some interest. Previously, we demonstrated that β1 stabilizes voltage sensor activation, such that activation occurs at more negative voltages with β1 present. This decreases the work that Ca2+ must do to open the channel and thereby increases the channel's apparent Ca2+ affinity without altering the real affinities of the channel's Ca2+ binding sites. To explain the full effect of β1 on the channel's Ca2+ sensitivity, however, we also proposed that there must be effects of β1 on Ca2+ binding. Here, to test this hypothesis, we have used high-resolution Ca2+ dose–response curves together with binding site–specific mutations to measure the effects of β1 on Ca2+ binding. We find that coexpression of β1 alters Ca2+ binding at both of the BKCa channel's two types of high-affinity Ca2+ binding sites, primarily increasing the affinity of the RCK1 sites when the channel is open and decreasing the affinity of the Ca2+ bowl sites when the channel is closed. Both of these modifications increase the difference in affinity between open and closed, such that Ca2+ binding at either site has a larger effect on channel opening when β1 is present.