Store-Operated Ca2+ Channels Contribute to the Generation of Ca2+ Waves in Interdental Cells in the Cochleae.

Cochlear calcium (Ca2+) waves are vital regulators of the cochlear development and establishment of hearing function. Inner supporting cells are believed to be the main region generating Ca2+ waves that work as internal stimuli to coordinate the development of hair cells and the mapping of neurons in the cochlea. However, Ca2+ waves in interdental cells (IDCs) that connect to inner supporting cells and spiral ganglion neurons are rarely observed and poorly understood. Herein, we reported the mechanism of IDC Ca2+ wave formation and propagation by developing a single-cell Ca2+ excitation technology, which can easily be accomplished using a two-photon microscope for simultaneous microscopy and femtosecond laser Ca2+ excitation in any target individual cell in fresh cochlear tissues. We demonstrated that the store-operated Ca2+ channels in IDCs are responsible for Ca2+ wave formation in these cells. The specific architecture of the IDCs determines the propagation of Ca2+ waves. Our results provide the mechanism of Ca2+ formation in IDCs and a controllable, precise, and noninvasive technology to excite local Ca2+ waves in the cochlea, with good potential for research on cochlear Ca2+ and hearing functions.

Neutrophil activation and periodontal tissue injury.

Neutrophilic polymorphonuclear leukocytes (PMNL) track, engage and eliminate foreign entities, including bacteria, fungi and subcellular particles. PMNL are the major host-cell line involved in the acute response during the early stages of infections, including those in the oral cavity. Rather short lived, they are among the fastest moving cells in the human body and travel great distances only to be immolated after encountering and neutralizing antigens. Although their role as the first line of host defense is well established, their role in chronic granulomatous inflammations, diseases and infections remains poorly understood, and many questions on the activation, motility, bactericidity and termination of PMNL in these conditions remain unanswered. This review aims to summarize our current understanding of the molecular mechanisms of PMNL activation and signaling events. Recent evidence indicates the presence of collateral tissue damage caused by poorly regulated PMNL pursuits of periodontal bacteria. Imbalances between the antigenic challenge and the primary host response may augment periodontal tissue breakdown. Thereafter, orchestrated regulation of the resolution of inflammation fails in the presence of a pathogenic periodontal biofilm.

Key components of store-operated Ca2+ entry in non-excitable cells.

Store-operated Ca(2+) entry (SOCE) is a ubiquitous Ca(2+) entry pathway in non-excitable cells. It is activated by the depletion of Ca(2+) from intracellular Ca(2+) stores, notably the endoplasmic reticulum (ER). In the past 9 years, it has been established that two key proteins, stromal interacting molecule 1 (STIM1) and Orai1, play critical roles in SOCE. STIM1 is a single-pass transmembrane protein located predominantly in the ER that serves as a Ca(2+) sensor within the ER, while Orai1 is a tetraspanning plasma membrane (PM) protein that functions as the pore-forming subunit of store-operated Ca(2+) channels. A decrease in the ER Ca(2+) concentration induces translocation of STIM1 into puncta close to the PM. STIM1 oligomers directly interact with Orai1 channels and activates them. This review summarizes the molecular basis of the interaction between STIM1 and Orai1 in SOCE. Further, we describe current findings on additional regulatory proteins, such as Ca(2+) release-activated Ca(2+) regulator 2A and septin, novel roles of STIM1, and modulation of SOCE by protein phosphorylation.

Calmodulin-like protein AtCML3 mediates dimerization of peroxisomal processing protease AtDEG15 and contributes to normal peroxisome metabolism.

Matrix enzymes are imported into peroxisomes and glyoxysomes, a subclass of peroxisomes involved in lipid mobilization. Two peroxisomal targeting signals (PTS), the C-terminal PTS1 and the N-terminal PTS2, mediate the translocation of proteins into the organelle. PTS2 processing upon import is conserved in higher eukaryotes, and in watermelon the glyoxysomal processing protease (GPP) was shown to catalyse PTS2 processing. GPP and its ortholog, the peroxisomal DEG protease from Arabidopsis thaliana (AtDEG15), belong to the Deg/HtrA family of ATP-independent serine proteases with Escherichia coli DegP as their prototype. GPP existes in monomeric and dimeric forms. Their equilibrium is shifted towards the monomer upon Ca(2+)-removal and towards the dimer upon Ca(2+)-addition, which is accompanied by a change in substrate specificity from a general protease (monomer) to the specific cleavage of the PTS2 (dimer). We describe the Ca(2+)/calmodulin (CaM) mediated dimerization of AtDEG15. Dimerization is mediated by the CaM-like protein AtCML3 as shown by yeast two and three hybrid analyses. The binding of AtCML3 occurs within the first 25 N-terminal amino acids of AtDEG15, a domain containing a predicted CaM-binding motif. Biochemical analysis of AtDEG15 deletion constructs in planta support the requirement of the CaM-binding domain for PTS2 processing. Phylogenetic analyses indicate that the CaM-binding site is conserved in peroxisomal processing proteases of higher plants (dicots, monocots) but not present in orthologs of animals or cellular slime molds. Despite normal PTS2 processing activity, an atcml3 mutant exhibited reduced 2,4-DB sensitivity, a phenotype previously reported for the atdeg15 mutant, indicating similarly impaired peroxisome metabolism.

Unraveling STIM2 function

The discovery of molecular players in capacitative calcium (Ca2+) entry, also referred to as store-operated Ca2+ entry (SOCE), supposed a great advance in the knowledge of cellular mechanisms of Ca2+ entry, which are essential for a broad range of cellular functions. The identification of STIM1 and STIM2 proteins as the sensors of Ca2+ stored in the endoplasmic reticulum unraveled the mechanism by which depletion of intracellular Ca2+ stores is communicated to store-operated Ca2+channels located in the plasma membrane, triggering the activation of SOCE and intracellular Ca2+-dependent signaling cascades. Initial studies suggested a dominant function of STIM1 in SOCE and SOCE-dependent cellular functions compared to STIM2, especially those that participate in immune responses. Consequently, most of the subsequent studies focused on STIM1. However, during the last years, STIM2 has been demonstrated to play a more relevant and complex function than initially reported, being even important to sustain normal life in mice. These studies have led to reconsider the role of STIM2 in SOCE and its relevance in cellular physiology. This review is intended to summarize and provide an overview of the current data available about this exciting isoform, STIM2, and its actual position together with STIM1 in the mechanism of SOCE (AU)

Calaxin drives sperm chemotaxis by Ca²âº-mediated direct modulation of a dynein motor.

Sperm chemotaxis occurs widely in animals and plants and plays an important role in the success of fertilization. Several studies have recently demonstrated that Ca(2+) influx through specific Ca(2+) channels is a prerequisite for sperm chemotactic movement. However, the regulator that modulates flagellar movement in response to Ca(2+) is unknown. Here we show that a neuronal calcium sensor, calaxin, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis. Calaxin inhibition resulted in significant loss of sperm chemotactic movement, despite normal increases in intracellular calcium concentration. Using a demembranated sperm model, we demonstrate that calaxin is essential for generation and propagation of Ca(2+)-induced asymmetric flagellar bending. An in vitro motility assay revealed that calaxin directly suppressed the velocity of microtubule sliding by outer-arm dynein at high Ca(2+) concentrations. This study describes the missing link between chemoattractant-mediated Ca(2+) signaling and motor-driven microtubule sliding during sperm chemotaxis.

Mechanisms of cellular synchronization in the vascular wall. Mechanisms of vasomotion.

Although the function of rhythmic contractions in the vascular wall - vasomotion - is still under debate, it has been suggested to play a significant role for tissue oxygen homeostasis and under pathological conditions where tissue perfusion is affected. Vasomotion has further been suggested to be important for blood pressure control and has been shown to be reduced in diabetes. Vasomotion is initiated by the coordinated activation of smooth muscle cells (SMCs) in the vascular wall leading to rhythmic contractions. We have suggested the model for generation of this rhythmic activity and have shown that vasomotion initiates via interaction between intracellular calcium released from the sarcoplasmic reticulum and changes in membrane potential. Rhythmic changes in intracellular calcium induce, under certain conditions (in the presence of sufficient concentration of cGMP), changes in membrane potential that lock the electrically-connected SMCs into phase. Synchronized depolarization induces synchronous calcium influx and thus produces rhythmic contraction of blood vessels. I have demonstrated and characterized a new chloride channel in vascular SMCs, which has properties necessary to coordinate SMCs in the vascular wall. Chloride channels have been investigated for many years but remained somewhat in the shadow of cation channels. We know now the molecular structures of some chloride channels, i.e. GABA receptors, "cystic fibrosis transmem-brane conductance regulator" (CFTR) and the ClC chloride channel family. There is one particular group of chloride channels, the calcium activated chloride channels (CaCCs), whose molecular structure is debated still. There are currently no pharmacological tools that activate or inhibit CaCCs with any significant selectivity. The existence of CaCCs in almost all cells in the body has been known for many years based on electrophysiological and other functional studies. CaCCs have been suggested to be important for regulation of membrane potential and cellular volume, as well as for body homeostasis. CaCCs are well characterized in vascular tissues but only at the functional level. The lack of their molecular structure makes it difficult to study the clinical significance of these channels. Based on patch clamp measurements of ion currents, I have previously characterized in SMCs a chloride current with unique properties. This chloride current activated by cGMP, has very high sensitivity to calcium and can be inhibited by low concentrations of zinc ions, while the traditional inhibitors of CaCCs affect this current only at very high concentrations. This cGMP-dependent, calcium-activated chloride current has a linear volt-age-dependence, which differs from previously characterized CaCCs, and it has characteristic anion permeability. This current has been detected in SMCs isolated from a number of different vascular beds but, importantly, it has not been detected in pulmonary arteries. Moreover, this current has been shown in SMCs isolated intestine indicating its broad distribution. Based on unique characteristics I have suggested that the cGMP-dependent calcium-activated chloride current can synchronize SMCs in the vascular wall and that bestrophin protein could be the molecular substrate for this current. Bestrophin has been characterized first as a gene in which mutations cause vitelliform macular dystrophy (VMD) or Best diseases. Based on heterologous expression it has been suggested that bestrophin is a chloride channel. This question is nevertheless controversial since caution should be taken in heterologous expression of calcium-activated chloride channel candidates. The presence of chloride channels in virtually all living cells is an essential problem as well as the dependence of ion channel properties on the complex interaction of many cellular proteins. I was the first who coupled the endogenous chloride current to one of four known bestrophin isoforms. PCR and Western blot studies on different blood vessels demonstrated the presence of bestrophin-3 protein with the exception of pulmonary arteries where the cGMP-dependent current is also absent). There was a strong indication that bestrophin-3 expression could be essential for the cGMP-dependent calcium-activated chloride current. To couple bestrophin-3 expression and this current I have used small interfering RNA (siRNA) technique to downregulate the expression of the candidate (bestrophin-3) and have studied the effect of this specific downregulation on chloride currents. I showed that bestrophin-3 expression is associated with the cGMP-dependent calcium-activated chloride current. This study does not tell us whether bestrophin-3 forms the channel or it is an essential subunit but the previous mutagenic experiments suggested the first possibility. Electrical communication between SMCs is essential for successful synchronization and depends on channels between the cells called gap junctions. The majority of cardiovascular diseases (e.g. hypertension and atherosclerosis) are associated with defects in intercellular communications or in gap junction regulation. The molecular mechanisms responsible for these defects are un-known because of lack of specific experimental tools. Our comprehensive study on the often used gap junction inhibitors heptanol and 18ß-glycyrrhetinic acid demonstrated unspecific effects of these drugs at the concentrations where they have no or little gap junctions effects. Other drugs, e.g. 18α-glycyrrhetinic acid and connexin-mimetic peptides are better to inhibit gap junctions but also have demonstrated unspecific effects. Previous studies suggested that channels and transporters in the cell membrane do not function independently but interact as functional units in the spatially restricted areas of the cell. I have demonstrated a close functional interaction between gap junctions and Na+,K+-ATPase, Na+/Ca2+-exchanger and ATP-dependent K+ channels in the spatially restricted manner. I have shown that inhibition of the ouabain-sensitive Na+, K+-ATPase inhibits calcium efflux by the Na+/Ca2+-exchanger and this leads to the local elevation of intracellular calcium and inhibition of intercellular communications. This explains the inhibitory action of ouabain on vasomotion. I have also found that the ATP-dependent K+ channel is an important player in this functional unit and this interaction is reciprocal, since K+ channel supplies Na+, K+-ATPase with K+ ions while the ATP-dependent K+ channel current also regulates the Na+, K+-ATPase. This dissertation is based on nine scientific publications where I have suggested the model for generation of vasomotion and characterized the essential elements of this model.

Distinct roles of four gelsolin-like domains of Caenorhabditis elegans gelsolin-like protein-1 in actin filament severing, barbed end capping, and phosphoinositide binding.

Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) is a new member of the gelsolin family of actin regulatory proteins [Klaavuniemi, T., Yamashiro, S., and Ono, S. (2008) J. Biol. Chem. 283, 26071-26080]. It is an unconventional gelsolin-related protein with four gelsolin-like (G) domains (G1-G4), unlike typical gelsolin-related proteins with three or six G domains. GSNL-1 severs actin filaments and caps the barbed end in a calcium-dependent manner similar to that of gelsolin. In contrast, GSNL-1 has properties different from those of gelsolin in that it remains bound to F-actin and does not nucleate actin polymerization. To understand the mechanism by which GSNL-1 regulates actin dynamics, we investigated the domain-function relationship of GSNL-1 by analyzing activities of truncated forms of GSNL-1. G1 and the linker between G1 and G2 were sufficient for actin filament severing, whereas G1 and G2 were required for barbed end capping. The actin severing activity of GSNL-1 was inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and a PIP2-sensitive domain was mapped to G1 and G2. At least two actin-binding sites were detected: a calcium-dependent G-actin-binding site in G1 and a calcium-independent G- and F-actin-binding site in G3 and G4. These results reveal both conserved and different utilization of G domains between C. elegans GSNL-1 and mammalian gelsolin for actin regulatory functions.

Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+ -induced glucagon exocytosis in pancreas.

Hormones such as glucagon are secreted by Ca(2+)-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic beta-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca(2+)-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca(2+) sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca(2+) sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca(2+)-triggered glucagon release from alpha-cells than insulin secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca(2+) sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca(2+) sensors in neurotransmitter, neuropeptide, and hormone secretion.

Caenorhabditis elegans gelsolin-like protein 1 is a novel actin filament-severing protein with four gelsolin-like repeats.

The gelsolin family of proteins is a major class of actin regulatory proteins that sever, cap, and nucleate actin filaments in a calcium-dependent manner and are involved in various cellular processes. Typically, gelsolin-related proteins have three or six repeats of gelsolin-like (G) domain, and each domain plays a distinct role in severing, capping, and nucleation. The Caenorhabditis elegans gelsolin-like protein-1 (gsnl-1) gene encodes an unconventional gelsolin-related protein with four G domains. Sequence alignment suggests that GSNL-1 lacks two G domains that are equivalent to fourth and fifth G domains of gelsolin. In vitro, GSNL-1 severed actin filaments and capped the barbed end in a calcium-dependent manner. However, unlike gelsolin, GSNL-1 remained bound to the side of F-actin with a submicromolar affinity and did not nucleate actin polymerization, although it bound to G-actin with high affinity. These results indicate that GSNL-1 is a novel member of the gelsolin family of actin regulatory proteins and provide new insight into functional diversity and evolution of gelsolin-related proteins.

Regulation of presynaptic Ca(V)2.1 channels by Ca2+ sensor proteins mediates short-term synaptic plasticity.

Short-term synaptic plasticity shapes the postsynaptic response to bursts of impulses and is crucial for encoding information in neurons, but the molecular mechanisms are unknown. Here we show that activity-dependent modulation of presynaptic Ca(V)2.1 channels mediated by neuronal Ca(2+) sensor proteins (CaS) induces synaptic plasticity in cultured superior cervical ganglion (SCG) neurons. A mutation of the IQ-like motif in the C terminus that blocks Ca(2+)/CaS-dependent facilitation of the P/Q-type Ca(2+) current markedly reduces facilitation of synaptic transmission. Deletion of the nearby calmodulin-binding domain, which inhibits CaS-dependent inactivation, substantially reduces depression of synaptic transmission. These results demonstrate that residual Ca(2+) in presynaptic terminals can act through CaS-dependent regulation of Ca(V)2.1 channels to induce short-term synaptic facilitation and rapid synaptic depression. Activity-dependent regulation of presynaptic Ca(V)2.1 channels by CaS proteins may therefore be a primary determinant of short-term synaptic plasticity and information-processing in the nervous system.

Targeting the sarcolemmal calcium pump: a potential novel strategy for the treatment of cardiovascular disease.

Intracellular calcium dynamics play a very important role in mediating contraction and signalling in cardiomyocytes and vascular smooth muscle cells. A number of calcium transporters have been identified that orchestrate a complex process of excitation-contraction coupling and molecular signalling. Despite the variability of the calcium transporters expressed in cardiomyocytes, most calcium channel blockers used therapeutically target the L-type calcium channel and exhibit antihypertensive and/or vasodilating activities. Recently, another calcium pump which is located in the sarcolemma has been shown to mediate cardiac contractility and vascular tone. Interestingly, this sarcolemmal calcium pump (also known as Plasma Membrane Calcium/calmodulin dependent ATPase or PMCA) exerts its function not by altering global calcium concentration, but by mediating signal transduction pathways. This review will discuss recent advances that support the key roles of PMCA as signalling molecule and the potential to target this calcium pump as a novel approach for the treatment of cardiovascular disease.

Propagated endothelial Ca2+ waves and arteriolar dilation in vivo: measurements in Cx40BAC GCaMP2 transgenic mice.

To study endothelial cell (EC)- specific Ca(2+) signaling in vivo we engineered transgenic mice in which the Ca(2+) sensor GCaMP2 is placed under control of endogenous connexin40 (Cx40) transcription regulatory elements within a bacterial artificial chromosome (BAC), resulting in high sensor expression in arterial ECs, atrial myocytes, and cardiac Purkinje fibers. High signal/noise Ca(2+) signals were obtained in Cx40(BAC)-GCaMP2 mice within the ventricular Purkinje cell network in vitro and in ECs of cremaster muscle arterioles in vivo. Microiontophoresis of acetylcholine (ACh) onto arterioles triggered a transient increase in EC Ca(2+) fluorescence that propagated along the arteriole with an initial velocity of approximately 116 microm/s (n=28) and decayed over distances up to 974 microm. The local rise in EC Ca(2+) was followed (delay, 830+/-60 ms; n=8) by vasodilation that conducted rapidly (mm/s), bidirectionally, and into branches for distances exceeding 1 mm. At intermediate distances (300 to 600 microm), rapidly-conducted vasodilation occurred without changing EC Ca(2+), and additional dilation occurred after arrival of a Ca(2+) wave. In contrast, focal delivery of sodium nitroprusside evoked similar local dilations without Ca(2+) signaling or conduction. We conclude that in vivo responses to ACh in arterioles consists of 2 phases: (1) a rapidly-conducted vasodilation initiated by a local rise in EC Ca(2+) but independent of EC Ca(2+) signaling at remote sites; and (2) a slower complementary dilation associated with a Ca(2+) wave that propagates along the endothelium.

Chloride-dependent intracellular pH regulation via extracellular calcium-sensing receptor in the medullary thick ascending limb of the mouse kidney.

The extracellular calcium-sensing receptor (CaSR) located in either luminal or basolateral cell membranes of various types of renal tubules including proximal tubules, Henle's loop and collecting ducts has been thought to play a fundamental role in electrolyte metabolism. To further identify the physiological roles of the CaSR, we examined the effects of Ca(2+) and calcimimetics neomycin (Neo), gentamicin and gadolinium chloride (Gd(3+)) on the intracellular pH (pHi) of in vitro microperfused mouse medullary thick ascending limb (mTAL) cells of Henle's loop, by loading the cells with fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein and measuring the ratio of fluorescence emission at 530 nm after exciting the dye at 490 and 440 nm. In a steady-state condition in Hepes-buffered solution, the pHi in the mTALs was 7.29 +/- 0.04 (n = 9). A concentration of 200 micromol/l Neo in the basolateral side decreased the pHi after 1 min by -0.13 +/- 0.02 (n = 34, p < 0.0001). The other calcimimetics showed similar effects on pHi, whereas none of these calcimimetics in the lumen affected pHi. Na(+) removal or the inhibition of Na(+) and proton transport with amiloride, bumetanide, or bafilomycin did not eliminate the effect of Neo on pHi. On the other hand, Cl(-) removal clearly eliminated the Neo-induced pHi decrease (-0.06 +/- 0.01 vs -0.00 +/- 0.05 in Cl(-) removal, n = 4, p < 0.003). Thus, we have demonstrated for the first time that the CaSR is involved in the regulation of the pHi in the mTAL and requires Cl(-) to exert its effect.