Point amino acid substitutions in Ca2+-binding centers of recoverin. III. Mutant with the fourth reconstructed Ca2+-binding site

Unlike wild type recoverin with only two (the second and the third) functioning Ca(2+)-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca(2+)-binding site. This site was reconstructed from the fourth potential Ca(2+)-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca(2+)-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca2+ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild type recoverin.     

Point amino acid substitutions in the Ca2+-binding sites of recoverin: II. The unusual behavior of the protein upon the binding of calcium ions

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca²⁺-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca²⁺ ions results in an increase in the mobility of the environment of Trp residues, in hydrophobicity, and in thermal stability (its thermal transition shifts by 15°C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca²⁺ are also characteristic of the -EF2 mutant of recoverin whose second Ca²⁺-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca²⁺-binding sites, respectively, are modified and damaged) are practically indifferent to the presence of calcium ions. For the communication I, see [1].     

Point amino acid substitutions in the Ca2+-binding centers of recoverin. I. Mechanism of successive filling of Ca2+-binding centers

The molecule of photoreceptor Ca(2+)-binding protein recoverin contains four potential Ca(2+)-binding sites of the EF-hand type, but only two of them (the second and the third) can actually bind calcium ions. We studied the interaction of Ca2+ with recoverin and its mutant forms containing point amino acid substitutions at the working Ca(2+)-binding sites by measuring the intrinsic protein fluorescence and found that the substitution of Gln for Glu residues chelating Ca2+ in one (the second or the third) or simultaneously in both (the second and the third) Ca(2+)-binding sites changes the affinity of the protein to Ca2+ ions in different ways. The Gln for Glu121 substitution in the third site and the simultaneous Gln substitutions in the second (for Glu85) and in the third (for Glu121) sites result in the complete loss of the capability of recoverin for a strong binding of Ca(2+)-ions. On the other hand, the Gln for Glu85 substitution only in the second site moderately affects its affinity to the cation. Hence, we assumed that recoverin successively binds Ca(2+)-ions: the second site is filled with the cation only after the third site has been filled. The binding constants for the third and the second Ca(2+)-binding sites of recoverin determined by spectrofluorimetric titration are 3.7 x 10(6) and 3.1 x 10(5) M-1, respectively.     

Single amino acid substitutions in the Ca2+-binding site of recoverin.II.The unusual behavior of the protein upon the binding of calcium ions

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca(2+)-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca2+ ions results in an increase in the mobility of the environment of Trp residues, in higher hydrophobicity, and in elevated thermal stability (its thermal transition shifts by 15 degrees C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca2+ are also characteristic of the -EF2 mutant of recoverin whose second Ca(2+)-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca(2+)-binding sites, respectively, are modified and damaged) are practically indifferent to calcium ions.     

Oligomerization of the Polycystin-2 C-terminal Tail and Effects on Its Ca2+-binding Properties

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca²⁺-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca²⁺-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca²⁺ bound, although TRP channels are believed to be tetramers. We found that there is only one Ca²⁺-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca²⁺ binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca²⁺ binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca²⁺-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca²⁺ binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca²⁺-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca²⁺-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca²⁺-binding proteins.     

Inhibition of the Ca2+-and phospholipid-dependent protein kinase by a novel Mr 17,000 Ca2+-binding protein

A novel M_r 17,000 Ca²⁺-binding protein isolated from bovine brain was found to be a potent inhibitor of the Ca²⁺- and phospholipid-dependent protein kinase (protein kinase C), also isolated from bovine brain. Halfmaximal inhibition by this calciprotein of the initial rate of phosphorylation of histone III-S by protein kinase C occurred at a calciprotein concentration of 2.2 μM under standard conditions. Comparison of the effects of a number of Ca²⁺-binding proteins on protein kinase C activity indicated that the M_r 17,000 Ca²⁺-binding protein was the most potent inhibitor, followed by the intestinal Ca²⁺-binding protein and calcineurin. Calmodulin, troponin C, S-100 protein and a M_r 21,000 Ca²⁺-binding protein of bovine brain were relatively weak inhibitors of protein kinase C. The inhibitory effect of the M_r 17,000 Ca²⁺-binding protein was apparently not due to its interaction with phospholipid or the basic protein substrate and therefore appears to be due to a direct effect on the protein kinase C. These observations suggest that the novel M_r 17,000 Ca²⁺-binding protein, and possibly other Ca²⁺-binding proteins, may play a physiological role in regulating the activity of protein kinase C.     

Roles of Two Ca2+-binding Domains in Regulation of the Cardiac Na+-Ca2+ Exchanger

We expressed full-length Na⁺-Ca²⁺ exchangers (NCXs) with mutations in two Ca²⁺-binding domains (CBD1 and CBD2) to determine the roles of the CBDs in Ca²⁺-dependent regulation of NCX. CBD1 has four Ca²⁺-binding sites, and mutation of residues Asp⁴²¹ and Glu⁴⁵¹, which primarily coordinate Ca²⁺ at sites 1 and 2, had little effect on regulation of NCX by Ca²⁺. In contrast, mutations at residues Glu³⁸⁵, Asp⁴⁴⁶, Asp⁴⁴⁷, and Asp⁵⁰⁰, which coordinate Ca²⁺ at sites 3 and 4 of CBD1, resulted in a drastic decrease in the apparent affinity of peak exchange current for regulatory Ca²⁺. Another mutant, M7, with 7 key residues of CBD1 replaced, showed a further decrease in apparent Ca²⁺ affinity but retained regulation, confirming a contribution of CBD2 to Ca²⁺ regulation. Addition of the mutation K585E (located in CBD2) into the M7 background induced a marked increase in Ca²⁺ affinity for both steady-state and peak currents. Also, we have shown previously that the CBD2 mutations E516L and E683V have no Ca²⁺-dependent regulation. We now demonstrate that introduction of a positive charge at these locations rescues Ca²⁺-dependent regulation. Finally, our data demonstrate that deletion of the unstructured loops between β-strands F and G of both CBDs does not alter the regulation of the exchanger by Ca²⁺, indicating that these segments are not important in regulation. Thus, CBD1 and CBD2 have distinct roles in Ca²⁺-dependent regulation of NCX. CBD1 determines the affinity of NCX for regulatory Ca²⁺, although CBD2 is also necessary for Ca²⁺-dependent regulation.     

Influence of myosin heavy chains on the Ca2+-binding properties of light chain, LC2

The association of myosin light chains with heavy chains, i.e. the intact oligomeric structure, profoundly affects the Ca²⁺-binding properties of the light chains. The Ca²⁺-binding affinity of the light chains is more than two magnitudes higher in the presence of heavy chains than in its absence. Modification of the reactive SH₂ thiol of myosin results in an alteration in the conformation of heavy chains of the molecule that influences the Ca²⁺-binding properties of light chains and generation of tension. When the SH₂ moiety is blocked with N-ethylmaleimide the influence of the heavy chains on the Ca²⁺-binding properties of light chain LC₂ is lost; under these conditions the Ca²⁺-binding affinity value of SH₂-N-ethylmaleimide-blocked myosin (3.3×10⁴m⁻¹) decreases to near that expressed with the dissociated light chain LC₂ (0.7×10⁴m⁻¹). Conversely, the presence of actin, nucleotides or modification of either the reactive lysyl residue or SH₂ thiol does not affect Ca²⁺ binding. The native secondary and tertiary structure of myosin seem to be required for Ca²⁺ binding; binding does not occur in the presence of 6m-urea with either native myosin or the dissociated light chains. With SH₂-N-ethylmaleimide-blocked myosin normal Ca²⁺- and (Mg²⁺+actin)-stimulated ATPase activities are expressed; however, there is a loss in K⁺-stimulated ATPase activity and the synthetic actomyosin threads of such myosin express no isometric tension. There are also variances in the binding of Ca²⁺ with alterations in pH values. In the absence of Ca²⁺/EGTA buffer the biphasic Ca²⁺-binding affinity of myosin is twice as high at pH7.4 (site one: 1.2×10⁶m⁻¹ and site two: 0.4×10⁶m⁻¹) as compared with values obtained at pH6.5 (site one: 0.64×10⁶m⁻¹ and site two: 0.2×10⁶m⁻¹). The Ca²⁺-binding affinity of light chain LC₂ and S₁, where the (S-1)–(S-2) junction was absent, were not influenced by changes in pH values. Both expressed a low Ca²⁺-binding affinity, approx. 0.7×10⁴m⁻¹, whereas heavy meromyosin, where both (S-1) and (S-2) myosin subfragments were present, expressed a Ca²⁺-binding affinity value similar to that of native myosin, but was not biphasic. However, it is important to point out than in preparation of S₁ myosin subfragment light chain LC₂ was lost and thus was added back to the purified S₁ fraction. Light chain LC₂ was not, however, added to the heavy meromyosin fraction because it was not lost during preparation of the heavy meromyosin subfragment. In conclusion, it appears that the (S-1)–(S-2) junction is needed for the positioning of light chain LC₂ and thus influences its essential conformation for Ca²⁺ binding.     

Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca²⁺-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca²⁺. Recent cross-linking studies have suggested that PLB binding and Ca²⁺ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca²⁺-ATPase, preventing formation of E1, the conformation that binds two Ca²⁺ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca²⁺ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca²⁺ pump. Seven SERCA2a mutants with amino acid substitutions at Ca²⁺-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca²⁺ on N30C-PLB cross-linking to Lys³²⁸ of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca²⁺-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca²⁺; however, all were phosphorylatable by P_i to form E2P. Ca²⁺ inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca²⁺-ATPase mutant was observed in the absence of Ca²⁺. Importantly, however, micromolar Ca²⁺ inhibited PLB cross-linking only to mutants retaining a functional Ca²⁺-binding site I. The dynamic equilibrium between Ca²⁺ pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca²⁺-binding site regulating the physical association between PLB and SERCA2a.     

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. Here, we report crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu⁵²⁰, which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, our structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.     

Decreased Ca2+-binding and Ca2+-ATPase activities in heart sarcolemma upon phospholipid methylation

Heart sarcolemma has been shown to possess three catalytic sites (I, II and III) for methyl transferase activity (Panagia V, Ganguly PK and Dhalla NS. Biochim Biophys Acta 792: 245–253, 1984). In this study we examined the effect of phosphatidylethanolamine N-methylation on ATP-independent Ca²⁺ binding and ATPase activities in isolated rat heart sarcolemma. Both low affinity (1.25 mM Ca²⁺) and high affinity (50 µM Ca²⁺) Ca²⁺ binding activities were decreased following incubation of sarcolemmal membranes with AdoMet under optimal conditions for site II and III. Similarly, Ca²⁺ ATPase activities measured at 1.25 mM and 4 mM Ca²⁺ were depressed by phospholipid N-methylation. S-adenosyl homocysteine, a specific inhibitor of phospholipid N-methylation, prevented the depression of low affinity Ca²⁺ binding and Ca²⁺ ATPase activities, whereas the methylation-induced effect on the high affinity Ca²⁺ binding was not influenced by this agent. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino group blocking agent, also prevented the methylation-induced inhibition of both Ca²⁺ binding and Ca²⁺ ATPase. A further decrease in Ca²⁺ binding and Ca²⁺ ATPase activities together with a marked increase in the intramembranal level of PC was seen when membranes were methylated under the site III conditions in the presence of phosphatidyldimethylethanolamine as exogenous substrate. There was no effect of phospholipid methylation on sarcolemmal Na⁺-K⁺ ATPase and Mg²⁺ ATPase activities. These results indicate a role of phospholipid N-methylation in the regulation of sarcolemmal Ca²⁺ ATPase and low affinity ATP-independent Ca²⁺ binding.     

Ca2+-dependent regulatory activity of recoverin in photoreceptor raft structures: The role of caveolin-1

Recoverin is a Ca²⁺-binding protein implicated in the Ca²⁺-dependent regulation of desensitization of visual receptor rhodopsin in vertebrate retinal rods. Here we report that Ca²⁺ sensitivity of recoverin regulating rhodopsin phosphorylation increases in the presence of the photoreceptor membranes enriched in raft structures. The observed effect is mediated by a key protein component of raft structures caveolin-1. The presence of recombinant fragment Phe⁸¹-Arg¹⁰¹ of the caveolin-1 cytoplasmic domain enhances Ca²⁺ affinity of recoverin, therefore affecting its Ca²⁺-dependent regulatory activity.     

Structural differences between Pb2+- and Ca2+-binding sites in proteins: implications with respect to toxicity

Pb²⁺ is known to displace physiologically-relevant metal ions in proteins. To investigate potential relationships between Pb²⁺/protein complexes and toxicity, data from the protein data bank were analyzed to compare structural properties of Pb²⁺- and Ca²⁺-binding sites. Results of this analysis reveal that the majority of Pb²⁺ sites (77.1%) involve 2-5 binding ligands, compared with 6 ± 2 for non-EF-Hand and 7 ± 1 for EF-Hand Ca²⁺-binding sites. The mean net negative charge by site (1.7) fell between values noted for non-EF-Hand (1 ± 1) and EF-Hand (3 ± 1). Oxygen is the dominant ligand for both Pb²⁺ and Ca²⁺, but Pb²⁺ binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) Ca²⁺-binding sites. A comparison of binding geometries where Pb²⁺ has replaced Ca²⁺ in calmodulin (CaM) and Zn²⁺ in 5-aminolaevulinic acid dehydratase (ALAD) revealed protein structural changes that appear to be unrelated to ionic displacement. Structural changes observed with CaM may be related to opportunistic binding of Pb²⁺ in regions of high electrostatic charge, whereas ALAD may bind multiple Pb²⁺ ions in the active site. These results suggest that Pb²⁺ adapts to structurally-diverse binding geometries and that opportunistic binding may play an active role in molecular metal toxicity.     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

Conformational changes of a Ca2+-binding domain of the Na+/Ca2+ exchanger monitored by FRET in transgenic zebrafish heart

The Na⁺/Ca²⁺ exchanger is the major Ca²⁺ extrusion mechanism in cardiac myocytes. The activity of the cardiac Na⁺/Ca²⁺ exchanger is dynamically regulated by intracellular Ca²⁺. Previous studies indicate that Ca²⁺ binding to a high-affinity Ca²⁺-binding domain (CBD1) in the large intracellular loop is involved in regulation. We generated transgenic zebrafish with cardiac-specific expression of CBD1 linked to yellow and cyan fluorescent protein. Ca²⁺ binding to CBD1 induces conformational changes, as detected by fluorescence resonance energy transfer. With this transgenic fish model, we were able to monitor conformational changes of the Ca²⁺ regulatory domain of Na⁺/Ca²⁺ exchanger in intact hearts. Treatment with the positive inotropic agents ouabain and isoproterenol increased both Ca²⁺ transients and Ca²⁺-induced changes in fluorescence resonance energy transfer. The results indicate that Ca²⁺ regulation of the Na⁺/Ca²⁺ exchanger domain CBD1 changes with inotropic state. The transgenic fish models will be useful to further characterize the regulatory properties of the Na⁺/Ca²⁺ exchanger in vivo. Ca²⁺-binding domain; sodium/calcium exchange; zebrafish; fluorescence resonance energy transfer     

Cytosolic Ca2+ and H+ buffers in green algae: A comment

Summary Recently Plieth et al. [Protoplasma (1997) 198: 107–124; 199: 223] gave a quantitative picture of the Ca²⁺ and H⁺ buffers in green algae which we would like to comment. In that paper a mechanistic model was derived which describes the relationship between cytosolic Ca²⁺ and H⁺ assuming that Ca²⁺ and H⁺ interact with the same binding site of a Ca²⁺-H⁺-exchange buffer. But the increase of the cytosolic free Ca²⁺ concentration observed upon acidification can alternatively be described by a co-operative (n=2) protonation of a Ca²⁺/H⁺-binding buffer pointing to an allosteric mechanism of Ca²⁺ liberation. Furthermore we present evidences that the cytosolic buffer capacities for H⁺ (90 mM/pH) and Ca²⁺ (20 mM/pCa) given for Eremosphaera viridis were overestimated by a factor of three and three orders of magnitude, respectively.Abbreviations [Ca²⁺]_c free cytosolic - Ca²⁺ concentration     

Equilibrium Dialysis Measurements of the Ca2+-Binding Properties of Recombinant Radish Vacuolar Ca2+-Binding Protein Expressed in Escherichia coli

Vacuoles of radish (Raphanus sativus) contained a Ca²⁺-binding protein (RVCaB) of 43 kDa. We investigated the Ca²⁺-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca²⁺-binding properties of the recombinant protein were examined by equilibrium dialysis with ⁴⁵Ca²⁺ and small dialysis buttons. The protein was estimated to bind 19Ca²⁺ ions per molecule with a K _d for Ca²⁺ of 3.4 mM. Ca²⁺ was bound to the protein even in the presence of high concentrations of Mg²⁺ or K⁺. The results suggested that the protein bound Ca²⁺ with high ion selectivity, high capacity, and low affinity.     

Divergence of Ca2+ selectivity and equilibrium Ca2+ blockade in a Ca2+ release-activated Ca2+ channel

Prevailing models postulate that high Ca²⁺ selectivity of Ca²⁺ release-activated Ca²⁺ (CRAC) channels arises from tight Ca²⁺ binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca²⁺ binding for Ca²⁺ selectivity in recombinant Orai3 channels, which function as highly Ca²⁺-selective channels when gated by the endoplasmic reticulum Ca²⁺ sensor STIM1 or as poorly Ca²⁺-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca²⁺ blocked Na⁺ currents in both gating modes with a similar inhibition constant (K_i; ∼25 µM). Thus, equilibrium binding as set by the K_i of Ca²⁺ blockade cannot explain the differing Ca²⁺ selectivity of the two gating modes. Unlike STIM1-gated channels, Ca²⁺ blockade in 2-APB–gated channels depended on the extracellular Na⁺ concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na⁺ pore occupancy. Moreover, the second-order rate constants of Ca²⁺ blockade were eightfold faster in 2-APB–gated channels than in STIM1-gated channels. A four-barrier, three–binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca²⁺ and Na⁺ to simulate the faster rate constants of 2-APB–gated channels qualitatively reproduces their low Ca²⁺ selectivity, suggesting that ion entry and exit rates strongly affect Ca²⁺ selectivity. Noise analysis indicated that the unitary Na⁺ conductance of 2-APB–gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (P_o; ∼0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high P_o state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca²⁺ binding and kinetic factors contribute to high Ca²⁺ selectivity in CRAC channels.     

Hold the door: hPMCA1/neuroplastin interactions regulate Ca2+-binding site accessibility

In a September 2018 paper published in Nature Communications, Gong et al. identified the domains through which human PMCA1 and neuroplastin (NPTN) interact. Upon binding, hPMCA1 TM domains separate T110 in TM1 and A370 in TM3 to reveal the Ca²⁺-binding site. Thus, NPTN is able to directly modulate the accessibility of cytosolic Ca²⁺ to PMCA.