Structure of human Na+/H+ exchanger NHE1 regulatory region in complex with calmodulin and Ca2+

The ubiquitous mammalian Na⁺/H⁺ exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration, and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion-translocating N-terminal membrane domain and acts as a scaffold for signaling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighboring regions of NHE1 in a strongly Ca²⁺-dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity toward alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1_CaMBR) in complex with CaM and Ca²⁺. The C- and N-lobes of CaM bind the first and second helix of NHE1_CaMBR, respectively. Both the NHE1 helices and the Ca²⁺-bound CaM are elongated, as confirmed by small angle x-ray scattering analysis. Our x-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca²⁺ regulates NHE1 activity.     

Changes in calmodulin immunocytochemical localization associated with capacitation and acrosomal exocytosis of ram spermatozoa

The aim of this study was to determine the localization of calmodulin (CaM) in ram sperm and the possible changes during in vitro capacitation (CA) and the ionophore-induced acrosome reaction (AR). Likewise, changes in intracellular calcium levels ([Ca²⁺]_i) were also analysed by using flow cytometry. CA was induced in vitro in a medium containing BSA, CaCl₂, NaHCO₃, and AR by the addition of the calcium ionophore A23187. The acrosomal status was assessed by the chlortetracycline-fluorescence (CTC) assay. Flow cytometry (FC) analyses were performed by loading samples with Fluo-3 AM, that emits fluorescence at a high [Ca²⁺]_i, combined with propidium iodide (PI) that allowed us to discriminate sperm with/without an integral plasma membrane both with high/low [Ca²⁺]_i. Immunocytochemistry localized CaM to the flagellum, and some sperm also contained CaM in the head (equatorial and post-acrosomal regions). CA and AR resulted in a slight increase in the post-acrosomal labelling. The treatment of sperm with increasing concentrations of two CaM antagonists, W7 and calmidazolium (CZ), accounted for an increase in capacitated and acrosome-reacted CTC-sperm patterns. CZ induced a significant reduction in the content of three protein tyrosine-phosphorylated bands of approximately of 30, 40 and 45 kDa. However, W7 showed no significant effect at any of the studied concentrations. Neither of them significantly influenced protein serine and threonine phosphorylation. FC analysis revealed that the main subpopulation in the control samples contained 70% of the total sperm with integral plasma membrane and a medium [Ca²⁺]_i. After CA, 67.1% of the sperm preserved an integral membrane with a higher [Ca²⁺]_i. After AR, only 7.2% of the total sperm preserved intact membranes with a very high [Ca²⁺]_i. These results imply that CaM appears to be involved in ram sperm capacitation, and both treatments increased its localization in the post-acrosomal region.     

Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa

Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca²⁺-dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca²⁺ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin-regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca²⁺ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin-binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca²⁺ mediator. In the present study, sperm calmodulin-binding proteins were identified by mass spectrometry after Ca²⁺-dependent biotinylated-calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca²⁺-dependent manner by calmodulin-conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm-egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.     

Gap junction regulation by calmodulin

Intracellular Ca²⁺ activated calmodulin (CaM) inhibits gap junction channels in the low nanomolar to high micromolar range of [Ca²⁺]_i. This regulation plays an essential role in numerous cellular processes that include hearing, lens transparency, and synchronized contractions of the heart. Previous studies have indicated that gap junction mediated cell-to-cell communication was inhibited by CaM antagonists. More recent evidence indicates a direct role of CaM in regulating several members of the connexin family. Since the intracellular loop and carboxyl termini of connexins are largely “invisible” in electron microscopy and X-ray crystallographic structures due to disorder in these domains, peptide models encompassing the putative CaM binding sites of several intracellular domains of connexins have been used to identify the Ca²⁺-dependent CaM binding sites of these proteins. This approach has been used to determine the CaM binding affinities of peptides derived from a number of different connexin-subfamilies.     

Phosphorylation of triton X-100 soluble and insoluble protein substrates in a demembranated/reactivated human sperm model

Sperm motility is a process which involves a cascade of events mediated by cAMP and Ca²⁺, cAMP in the initiation of flagellar movement, and Ca²⁺ in the regulation of beat asymmetry, and it has been suggested that these two messengers act through phosphorylation/dephosphorylation of axonemal proteins. Only a few studies on human sperm protein phosphorylation have been reported and no relation of this process with motility or other function has been established. In the present study, phosphorylation of human sperm proteins was performed using detergent-demembranated spermatozoa, in which motility is reactivated by the addition of ATP. This system allows direct accessibility of intracellular kinases to [³²P]-γATP and allows some relation between protein phosphorylation and flagellar movements. After electrophoresis and autoradiography, numerous phosphoproteins were detected. Phosphorylation of 2 proteins (36 and 51 kDa) was stimulated by cAMP in a concentration-dependent manner, and this increase was prevented by inhibitors of cAMP-dependent protein kinase. In order to characterize phosphoproteins originating from the cytoskeleton or axoneme, detergent extracted spermatozoa were also subjected to phosphorylation. Three major phosphorylated proteins (14.8, 15.3, and 16.2 kDa) were detected, the first two expressing cAMP-dependency according to their cAMP concentration-dependent increase in phosphorylation and the reversal of this effect by inhibitors of cAMP-dependent protein kinase. Proteins phosphorylation during the reactivation of demembranated spermatozoa previously immobilized H₂O₂, xanthine + xanthine oxidase-generated reactive oxygen species, or the oxidative phosphorylation uncoupler rotenone, revealed increases in cAMP-independent phosphorylation of proteins of 16.2, 46, and 93 kDa. These results documenting human sperm phosphoproteins form a base for further studies on the role of protein phosphorylation in sperm functions. © 1996 Wiley-Liss, Inc.     

Changes in Calcium/Calmodulin Level and Mitochondrial Membrane Potential in Cochlear Neurons of Newborn Mice Infected with Murine Cytomegalovirus

We sought to elucidate the pathogenesis of hearing loss in newborns due to congenital cytomegalovirus. We used the model of murine cytomegalovirus (MCMV) infection and evaluated concentrations of free calcium, calmodulin levels, and mitochondrial membrane potential in cochlear neurons of infected newborn mice. MCMV infection was established by intracranial inoculation of newborn mice with viral suspension (20 μl of MCMV TCID₅₀—10⁴ IU/0.1 ml); the mice in control group were injected 0.9 % NaCl. Concentration of intracellular free calcium concentration ([Ca²⁺] _i ), mitochondrial membrane potential, and the mRNA level of calmodulin (CaM) in the cochlear neurons were evaluated, when the mice were 1 month old. Compared with control group, intracellular [Ca²⁺] _i and CaM mRNA levels significantly (p < 0.05; both comparisons) increased, while the mitochondrial membrane potential significantly (p < 0.05) decreased in the MCMV-infected group. In conclusion, alteration of [Ca²⁺] _i and CaM levels and mitochondrial membrane potentials in cochlear neurons may be the pathological basis of sensorineural hearing loss associated with MCMV infection.     

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca²⁺ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca ²⁺ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca ²⁺ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca ²⁺ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca ²⁺ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca ²⁺ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH ₄Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca ²⁺ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.     

Cytoplasm calcium-binding proteins of germ cells and embryos of the sea urchin

Synchronous, demonstrative, easily reproducible fertilization with the following embryonic development makes the process in the sea urchin extremely attractive for studying many biological enigmas. In particular, germ and embryonic cells of the sea urchin present a wide opportunity for investigating different associated phenomena launched by an increase in concentration of Ca²⁺ in cells ([Ca²⁺]_i).Ca²⁺ ions participate in the activation of diverse processes of respiration and sperm motility (Shapiro et al., 1990; Brokaw, 1991), chemotaxis of spermatozoa to components of the egg jelly (Ward et al., 1985), acrosomal reaction (Trimmer et al., 1986; Shapiro et al., 1990), cortical reaction, formation of the fertilization membrane (Sasaki, 1984; Sardet and Chang, 1987), cellular division in the embryo (Poenie et al., 1985; Silver, 1986; Whitaker and Patel, 1990), their adhesion (McClay and Matranga, 1986), differentiation and formation of spicules (Mitsunaga et al., 1988) and metamorphosis (Carpenter et al., 1984).The present review combines information on the function of calcium-binding proteins and their targets, calmodulin regulation of NAD-kinase, exocytosis of cortical granules, Ca²⁺- and calmodulin-dependent protein phosphatase, Ca²⁺-dependent protein phosphorylation, regulation of ion-exchanger in the germ and embryonic cells as well as Ca²⁺- and calmodulin control of sperm motility in sea urchins.     

CASK interacts with PMCA4b and JAM-A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility

Deletion of the highly conserved gene for the major Ca²⁺ efflux pump, Plasma membrane calcium/calmodulin‐dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild‐type (WT), Junctional adhesion molecule‐A (Jam‐A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P < 0.001) ATP levels, significantly (P < 0.001) greater cytosolic Ca²⁺ concentration ([Ca²⁺]_c) and ～10‐fold higher mitochondrial sequestration, indicating Ca²⁺ overload. Investigating the mechanism involved, we used co‐immunoprecipitation studies to show that CASK (Ca²⁺/calmodulin‐dependent serine kinase), identified for the first time on the sperm flagellum where it co‐localizes with both PMCA4b and JAM‐A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non‐synergistically with each of these molecules via its single PDZ (PDS‐95/Dlg/ZO‐1) domain to either inhibit or promote efflux. In the absence of CASK–JAM‐A interaction in Jam‐A null sperm, CASK–PMCA4b interaction is increased, resulting in inhibition of PMCA4b's enzymatic activity, consequent Ca²⁺ accumulation, and a ～6‐fold over‐expression of constitutively ATP‐utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM‐A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca²⁺ homeostasis in sperm is maintained by the relative ratios of CASK–PMCA4b and CASK–JAM‐A interactions. J. Cell. Physiol. 227: 3138–3150, 2012. © 2011 Wiley Periodicals, Inc.     

Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short‐term synaptic plasticity

Ca²⁺ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca²⁺–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca²⁺ signals. We solved the structure of Ca²⁺₄–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca²⁺₂–CaM complex. The Ca²⁺ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca²⁺–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca²⁺]_i, whereas the N‐module acts as a sensor at micromolar [Ca²⁺]_i. This Ca²⁺/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca²⁺‐dependent modulation of short‐term synaptic plasticity.     

Mitochondrial movement during the ascidian sperm reaction

The ascidian sperm reaction, Which involves swelling, migration, and loss of the single large mitochondrion, can be triggered in vitro by raising the seawater pH to 9.3 or lowering Na⁺ to 20 mM, but only if the sperm are allowed to attach to a suitable Substate. Mitochondrial translocation does not usually occur in the absence of sperm attachment. Extracellular Ca²⁺ is necessary for triggering the reaction with low Na⁺ but not high pH; however, the intrecellular Ca²⁺ blocker, TMB-8, inhibits high pH-induced mitochondrial movement in the absence of extracellular Ca²⁺. After swelling, the mitochondrion fluoresces in the presence of chlortetracycline, suggesting that Ca²⁺ becomes membranebound after activation. Elevated cAMP and theophylline both inhibit mitochondrial move ment but not sperm motility. The antiactin drug cytochalasin B(10μM) and the calmodulinblocking drugs TFP (1 μM) and W-13 (10 μM) block mitochondrial movement, suggesting roles for actin and calmodulin in mitochondrial movement. A model is proposed relating intracellular alkalinization, Ca²⁺ influx, actin, myosin, and calmodulin in mitochondrial translocation.     

Properties of a Novel pH-dependent Ca2+ Permeation Pathway Present in Male Germ Cells with Possible Roles in Spermatogenesis and Mature Sperm Function

Rises of intracellular Ca²⁺ ([Ca²⁺]_i) are key signals for cell division, differentiation, and maturation. Similarly, they are likely to be important for the unique processes of meiosis and spermatogenesis, carried out exclusively by male germ cells. In addition, elevations of [Ca²⁺]_i and intracellular pH (pH_i) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca²⁺ channels modulated by pH_i in the origin of these Ca²⁺ elevations, but their nature remains unexplored, in part because work in individual spermatozoa are hampered by formidable experimental difficulties. Recently, late spermatogenic cells have emerged as a model system for studying aspects relevant for sperm physiology, such as plasmalemmal ion fluxes. Here we describe the first study on the influence of controlled intracellular alkalinization on [Ca²⁺]_i on identified spermatogenic cells from mouse adult testes. In BCECF [(2′,7′)-bis(carboxymethyl)- (5,6)-carboxyfluorescein]-AM-loaded spermatogenic cells, a brief (30–60 s) application of 25 mM NH₄Cl increased pH_i by ∼1.3 U from a resting pH_i ∼6.65. A steady pH_i plateau was maintained during NH₄Cl application, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an initial [Ca²⁺]_i drop followed by a two- to threefold rise. Maneuvers that inhibit either Ca²⁺ influx or intracellular Ca²⁺ release demonstrated that the majority of the Ca²⁺ rise results from plasma membrane Ca²⁺ influx, although a small component likely to result from intracellular Ca²⁺ release was occasionally observed. Ca²⁺ transients potentiated with repeated NH₄Cl applications, gradually obliterating the initial [Ca²⁺]_i drop. The pH-sensitive Ca²⁺ permeation pathway allows the passage of other divalents (Sr²⁺, Ba²⁺, and Mn²⁺) and is blocked by inorganic Ca²⁺ channel blockers (Ni²⁺ and Cd²⁺), but not by the organic blocker nifedipine. The magnitude of these Ca²⁺ transients increased as maturation advanced, with the largest responses being recorded in testicular sperm. By extrapolation, these findings suggest that the pH-dependent Ca²⁺ influx pathway could play significant roles in mature sperm physiology. Its pharmacology and ion selectivity suggests that it corresponds to an ion channel different from the voltage-gated T-type Ca²⁺ channel also present in spermatogenic cells. We postulate that the Ca²⁺ permeation pathway regulated by pH_i, if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca²⁺ influx required for initiating the acrosome reaction and perhaps other important sperm functions.     

Ca regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca²⁺ ([Ca²⁺]_i) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca²⁺]_i was in the 500 nM range but the responses disappeared with larger [Ca²⁺]_i transients. Ca²⁺-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca²⁺-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca²⁺]_i triggers hemichannel opening, not by a direct action of Ca²⁺ on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca²⁺]_i and acting to restrain cellular ATP loss.     

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pH_i) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na⁺-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pH_i is more acidic than the external medium (pH_o = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na⁺ to sperm suspended in Na⁺-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pH_i by 0.4–0.5 pH units. That this pH_i change is the causal trigger of motility was suggested by experiments using NH₄Cl and nigericin, which increased the pH_i and resulted in activation of motility in the absence of Na⁺. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pH_i was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca²⁺ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pH_i by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca²⁺ uptake. Either mitochondrial agents or the removal of external Ca²⁺ could also block this pH_i change suggesting a similar mechanism is involved.     

The individual N- and C-lobes of calmodulin tether to the Cav1.2 channel and rescue the channel activity from run-down in ventricular myocytes of guinea-pig heart

The present study examined the binding of the individual N- and C-lobes of calmodulin (CaM) to Cav1.2 at different Ca²⁺ concentration ([Ca²⁺]) from ≈ free to 2 mM, and found that they may bind to Cav1.2 Ca²⁺-dependently. In particular, using the patch-clamp technique, we confirmed that the N- or C-lobes can rescue the basal activity of Cav1.2 from run-down, demonstrating the functional relevance of the individual lobes. The data imply that at resting [Ca²⁺], CaM may tether to the channel with its single lobe, leading to multiple CaM molecule binding to increase the grade of Ca²⁺-dependent regulation of Cav1.2.     

Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation

We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPβS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca²⁺-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca²⁺ induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca²⁺-containing but not in a Ca²⁺-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y₂ receptors and PI3K/Akt pathway activation involving Ca²⁺, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca²⁺ influx/CaM pathway.     

Zn induces hyperpolarization by activation of a K channel and increases intracellular Ca and pH in sea urchin spermatozoa

Zinc (Zn²⁺) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn²⁺ was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pH_i). In this study we found that submicromolar concentrations of free Zn²⁺ change membrane potential (Em) and increase the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn²⁺ response in sperm of this species mainly involves an Em hyperpolarization caused by K⁺ channel activation. The pharmacological profile of the Zn²⁺-induced hyperpolarization indicates that the cGMP-gated K⁺ selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn²⁺. Considering that Zn²⁺ also induces [Ca²⁺]_i fluctuations, our observations suggest that Zn²⁺ activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn²⁺ in male gamete function.     

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca²⁺–CaM, Ca²⁺–CaM–PDE1A, and Ca²⁺–CaM–MLCK complexes, CPZ apparent dissociation constants (K_ds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC K_ds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca²⁺–hCaM M124C–mBBr quenched the fluorescence (K_d = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca²⁺–hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca²⁺–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca²⁺–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd.     

Calcium Dynamics During Physiological Acidification in Xenopus Oocyte

Interplays between intracellular pH (pHi) and calcium ([Ca²⁺]_i) variations remain unclear, though both proton and calcium homeostasis changes accompany physiological events such as Xenopus laevis oocyte maturation. In this report, we used NH₄Cl and changes of extracellular pH (pHe) to acidify the cytosol in a physiological range. In oocytes voltage-clamped at −80 mV, NH₄Cl triggered an inward current, the main component of which is a Ca²⁺-dependent chloride current. Calcium imaging confirmed that NH₄Cl provoked a [Ca²⁺]_i increase. The mobilized sources of calcium were discriminated using the triple-step protocol as a means to follow both the calcium-activated chloride currents (ICl-Ca) and the hyperpolarization- and acid-activated nonselective cation current (I_In). These currents were stimulated during external addition of NH₄Cl. This upregulation was abolished by BAPTA-AM, caffeine and heparin. By both buffering pHi changes with MOPS and by inhibiting calcium influx with lanthanum, intracellular acidification, initiated by NH₄Cl and extracellular acidic medium, was shown to trigger a [Ca²⁺]_i increase through both calcium release and calcium influx. The calcium pathways triggered by pHe changes are similar to those activated by NH₄Cl, thus suggesting that there is a robust signaling mechanism allowing the cell to adjust to variable environmental conditions.