Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.     

Inactivation of L-type calcium channels in cardiomyocytes. Experimental and theoretical approaches

The L-type calcium current (ICa) plays an important role in excitation-contraction coupling of heart cells. It is critical for forming the major trigger for Ca(2+)-induced Ca(2+) release from the sarcoplasmic reticulum and hence its feedback regulation is of fundamental biological significance. The channel inactivation sharpens the kinetics and temporal precision of the Ca(2+) signals so that it prevents longer-term increases in free intracellular Ca(2+) concentration. Cardiac L-type Ca(2+) channels are known to inactivate through voltage- and Ca(2+)-dependent mechanisms. Pure voltage-dependent inactivation has a much slower time course of development than Ca(2+)-dependent inactivation and plays minor role in inhibition of Ca(2+) influx into the cell. The major determinant of the inactivation kinetics of Ca(2+) current during depolarization is Ca(2+)-dependent mechanisms. Furthermore, it is possible to distinguish two phases in Ca(2+)-dependent inactivation of calcium current: a slow phase that depends on Ca(2+) flow through the channels (Ca(2+) current-dependent inactivation) and a fast one that depends on Ca(2+) released from the sarcoplasmic reticulum (Ca(2+) release-dependent inactivation). Although both Ca(2+) released from the SR and Ca(2+) permeating channels play a role, SR-released Ca(2+) is the most effective inactivation mechanism in inhibition of Ca(2+) entry through the channel.     

Voltage- and calcium-dependent inactivation of calcium channels in Lymnaea neurons.

Ca(2+) channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca(2+) buffer (5 mM EGTA), the inactivation of these Ca(2+) channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca(2+) influx, or reduced by higher levels of intracellular Ca(2+) buffering. Inactivation measured under these conditions, despite being independent of Ca(2+) influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca(2+) buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca(2+)-dependent inactivation does not increase linearly with Ca(2+) influx, but saturates for relatively small amounts of Ca(2+) influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca(2+)-dependent inactivation suggests that the Ca(2+) binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca(2+) channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca(2+) channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca(2+) channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca(2+)-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca(2+) channels.     

Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains

The Ca(2+)-dependent oligomerization activity of the second C2 (C2B) domain of synaptotagmin I (Syt I) has been hypothesized to regulate neurotransmitter release. We previously showed that the cytoplasmic domains of several other Syt isoforms also show Ca(2+)-dependent oligomerization activity (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the involvement of their C2 domains in Ca(2+)-dependent oligomerization. In this study, we analyzed the Ca(2+)-dependent oligomerization properties of the first (C2A) and the second C2 (C2B) domains of Syt VII. Unlike Syt I, both C2 domains of Syt VII contribute to Ca(2+)-dependent homo- and hetero-oligomerization with other isoforms. For instance, the Syt VII C2A domain Ca(2+)-dependently binds itself and the C2A domain of Syt VI but not its C2B domain, whereas the Syt VII C2B domain Ca(2+)-dependently binds itself and the C2B domain of Syt II but not its C2A domain. In addition, we showed by gel filtration that a single Syt VII C2 domain is sufficient to form a Ca(2+)-dependent multimer of very high molecular weight. Because of this "two handed" structure, the Syt VII cytoplasmic domain has been found to show the strongest Ca(2+)-dependent multimerization activity in the Syt family. We also identified Asn-328 in the C2B domain as a crucial residue for the efficient Ca(2+)-dependent switch for multimerization by site-directed mutagenesis. Our results suggest that Syt VII is a specific isoform that can cluster different Syt isoforms with two hands in response to Ca(2+).     

Calcium ion-dependent p-nitrophenyl phosphate phosphatase activity and calcium ion-dependent adenosine triphosphatase activity from human erythrocyte membranes

In the presence of ATP and of Mg(2+), human erythrocyte membranes show a phosphatase activity towards p-nitrophenyl phosphate which is activated by low concentrations of Ca(2+). The effect of Ca(2+) is strongly enhanced if either K(+) or Na(+) is also present. Activation of the p-nitrophenyl phosphate phosphatase by Ca(2+) reaches a half-maximum at about 8mum-Ca(2+) and is apparent only when the ion has access to the inner surface of the cell membrane. Ca(2+)-dependent phosphatase activity can only be observed if ATP is at the inner surface of the cell membrane, and the presence of ATP seems to be absolutely necessary, since either its removal or its replacement by other nucleoside triphosphates abolishes the activating effect of Ca(2+). The properties of the (ATP+Ca(2+))-dependent phosphatase are very similar to those of the Ca(2+)-dependent ATPase (adenosine triphosphatase), also present in erythrocyte membranes, which probably is involved in Ca(2+) transport in erythrocytes. The similarities suggest that both activities may be properties of the same molecular system. This view is further supported by the fact that p-nitrophenyl phosphate inhibits to a similar extent Ca(2+)-dependent ATPase activity and ATP-dependent Ca(2+) extrusion from erythrocytes.     

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.     

Identification and characterization of "basal" Ca2+-independent Mg2+-dependent ATP-hydrolase enzymatic activity in smooth muscle cell plasma membrane fractions

It is shown, that for correct definition of "basal" Ca(2+)-independent Mg(2+)-dependent ATPase ac-activity (10-13 mmol Pi/hour on 1 mg of protein) in a fraction of uterus smooth muscle cell plasma membranes is necessary to use in medium without calcium of an incubation not only EGTA and digitonin--of the factor of infringement in activity by this subcellular structure, but inhibitors of others Mg(2+)-dependent ATP-hydrolyse enzymatic systems localized as in plasma membrane (Na+, K(+)-ATPase) and in others subcellular frames, first of all, in mitochondria (Mg(2+)-ATPase) and endoplasmic reticulum (transport Ca2+, Mg(2+)-ATPase). In the case of a sacolemal fraction of a smooth muscle the contribution of others Mg(2+)-dependent ATP-hydrolyse systems in a common enzymatic hydrolysis ATP, which unconnected to functioning "basal" Ca(2+)-independent Mg(2+)-dependent ATPase, is very appreciable and achieves 35%. The researches, carried out in the frameworks of definition of initial velocity of enzymatic reaction, have enabled to define its some properties--cationic and anionic specificity, and also sensitivity to action of some inhibitors. It has appeared, that the "basal" Ca(2+)-independent Mg(2+)-dependent ATP-hydrolyse reaction is nonspecific rather both in relation to cations of divalent metals Me2+, and cations of monovalent metals and anions, which were utilized for support of ionic strength. The cations La--antagonist of cations Ca--practically did not influence enzymatic activity. The non-specific inhibitors transport of ATPases--p-chloromercuribenzoate, o-vanadate and eosine Y with a various degree of efficiency inhibited "basal" Ca(2+)-independent Mg(2+)-dependent ATP-hydrolyse reaction. On the basis of the analysis of the own and literary data the conclusion is made that "basal" Ca(2+)-independent Mg(2+)-dependent ATPase of a smooth muscle cell plasma membrane is considerably less sensitive to action of nonspecific inhibitors of the Ca(2+)-transporting systems, than these systems.     

Physiological characterization of the Na(+)/Ca(2+) exchanger (NCX) in hepatopancreatic and antennal gland basolateral membrane vesicles isolated from the freshwater crayfish Procambarus clarkii

The purpose of this study was to physiologically characterize the basolateral Na(+)/Ca(2+) exchanger (NCX) in basolateral membrane vesicles (BLMVs) of hepatopancreas and antennal gland of intermolt crayfish. Conditions were optimized to measure Na(+)-dependent Ca(2+) uptake and retention in the BLMV including use of intravesicular (IV) oxalate and measuring initial uptake rates at 20 s. Na(+)-dependent Ca(2+) uptake rate into BLMV was temperature insensitive. Na(+)-dependent Ca(2+) uptake rate was dependent upon free Ca(2+) with saturable Michaelis-Menten kinetics determined as follows: hepatopancreas, maximal uptake rate (J(max))=2.45 nmol/mg per min, concentration at which carrier operates at half-maximal uptake rate (K(m))=0.69 microM Ca(2+); antennal gland, J(max)=13.2 nmol/mg per min, K(m)=0.59 microM Ca(2+). The two vesicle populations exhibited different sensitivity to putative NCX inhibitors. Benzamil had no effect on Na(+)-dependent Ca(2+) uptake rate in hepatopancreas; in antennal gland it was inhibitory at concentrations up to 30 microM and was stimulatory at higher concentrations. Conversely the inhibitor quinacrine was inhibitory at 10 microM in hepatopancreas and was stimulatory at 1000 microM; meanwhile it was ineffective in antennal gland BLMV. Short circuiting the BLMV had no effect on Na(+)-dependent Ca(2+) uptake rate suggesting that the process may be electroneutral. Compared with another prominent basolateral transporter in hepatopancreas the plasma membrane Ca(2+) ATPase (PMCA), the NCX has 70-fold greater J(max) (at comparable temperature) and a lower affinity. In antennal gland the NCX has 40-fold greater J(max) and a lower affinity. In hepatopancreas and antennal gland BLMV NCX appears to determine the rate of basolateral Ca(2+) efflux in intermolt.     

Calcium-dependent and calcium-independent gelatinolytic proteinase activities of the rat ventral prostate and its secretion: characterization and effect of castration and testosterone treatment

Calcium-dependent and calcium-independent proteinase activities were detected in extracts of rat ventral prostate and its secretion by use of gelatin-containing SDS-PAGE zymography. Ca(2+)-independent proteinase activities of 22, 26, and 73-79 kDa and Ca(2+)-dependent activities of 58, 63, and 66 kDa were found in the adult gland. The 26- (most intense activity of gland) and 22-kDa activities were present in secretion and were not expressed in the undifferentiated gland of the 10-day-old animal. The Ca(2+)-dependent activities were also present in the secretion, where the 63-kDa form was more prominently expressed than the 58- and 66-kDa bands. The Ca(2+)-dependent and Ca(2+)-independent proteinase activities both responded to a broad range of pH values in the incubation media. The 73-79-kDa Ca(2+)-independent activities were sensitive to benzamidine and the Ca(2+)-dependent activities were inhibited by EDTA and EGTA. Both Ca(2+)-dependent and Ca(2+)-independent proteinase activities responded to androgenic manipulations. Castration was followed by the appearance of a 35-kDa Ca(2+)-independent proteinase (at 2 days) and a 43-kDa Ca(2+)-dependent proteinase (at 4 days). In the Ca(2+)-independent proteinase group, the 73-79-kDa activities were increased somewhat and the 22- and 26-kDa activities decreased after castration. The Ca(2+)-dependent proteinases of 58, 63, and 66 kDa increased in activity with castration, but activity of the 58-kDa form decreased again at 7 days after castration. Treatment of animals upon castration for 4 days with hydrocortisone prevented these changes in proteinase activities whereas treatment with actinomycin D or tranexamic acid did not. Testosterone propionate replacement therapy of rats castrated for 16 days stimulated the activities of the 22- and 26-kDa and 73-79-kDa Ca(2+)-independent and the 58- and 63-kDa Ca(2+)-dependent proteinases with 4 days of therapy. The activities of the 35-kDa Ca(2+)-independent and the 43-kDa Ca(2+)-dependent proteinases were repressed with 8 days of testosterone treatment. Thus, individual proteinases show differential changes in activity during development and in response to androgenic manipulation: this suggests that in addition to proteinases which are secreted, others may be involved in intracellular functions or in mediating tissue organization changes.     

Molecular mechanism of the inhibitory effect of cobalt ion on thermolysin activity and the suppressive effect of calcium ion on the cobalt ion-dependent inactivation of thermolysin

Thermolysin activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA) and FA-l-leucyl-l-alanine amide (FALAA) was examined at various Co(2+) and Ca(2+) concentrations. It decreased to 28% with increasing [Co(2+)] up to 18 mM. The Co(2+)-dependent inactivation was in part suppressed by adding Ca(2+) ion up to 0.5 mM, but 33% of the activity remained to be inactivated even with a sufficient concentration of Ca(2+) (>0.5 mM). The Co(2+)-dependent inactivation was shown to be composed of Ca(2+)-sensitive and Ca(2+)-insensitive parts. In the latter part which is observed at [Ca(2+)] >0.5 mM, Co(2+) plays as a competitive inhibitor. On the other hand, the Co(2+)-dependent inactivation in the Ca(2+)-sensitive part observed at [Ca(2+)] <0.5 mM proceeds time-dependently following second-order kinetics, and the time-course is in good agreement with that of decrease in the thermolysin band due to autolysis in SDS-PAGE. This indicates that Co(2+) accelerates the autolysis. Here, we describe the co-regulation of thermolysin activity by Co(2+) and Ca(2+) ions and propose a molecular mechanism for the inhibition of thermolysin by Co(2+) and suppressive effect of Ca(2+) on the Co(2+)-dependent inhibition. Co(2+) ion inhibits thermolysin activity not only as a competitive inhibitor but also promoting the autolysis.     

A key role for a 145-kDa cytosolic protein in the stimulation of Ca(2+)-dependent secretion by protein kinase C.

Cytoplasmic Ca2+ is a major regulator of exocytosis in secretory cells; however, the Ca(2+)-dependent mechanisms that trigger secretion have not been elucidated. Protein kinase C (PKC) has been proposed to be an important Ca(2+)-dependent component of this regulation; however, the effects of this enzyme on the exocytotic apparatus have not been identified. We developed a PKC-deficient, semi-intact PC12 cell system in which direct stimulatory effects of purified PKC on Ca(2+)-dependent norepinephrine secretion were studied. The reconstitution of optimal Ca(2+)-activated norepinephrine secretion by semi-intact PC12 cells required the addition of MgATP and cytosolic proteins. PKC-deficient cytosol exhibited reduced reconstituting activity that was fully restored by the addition of purified PKC. The restoration of Ca(2+)-dependent norepinephrine secretion by PKC required the presence of other proteins in the cytosol, in particular, a high molecular weight protein. The high molecular weight protein was identified as p145, a recently characterized 145-kDa brain protein. The addition of PKC enhanced phosphorylation of p145 under conditions of fully reconstituted Ca(2+)-activated norepinephrine secretion. The results indicate that 1) PKC is neither necessary nor sufficient for Ca(2+)-activated secretion, whereas other cytosolic proteins are required; and 2) the stimulation of Ca(2+)-activated secretion by PKC is dependent upon cytosolic proteins such as p145 and may be largely mediated through the phosphorylation of p145.     

Modification of annexin II expression in PC12 cell lines does not affect Ca(2+)-dependent exocytosis.

The Ca2+/phospholipid/cytoskeletal-binding protein annexin II has been proposed to play an important role in Ca(2+)-dependent exocytosis; however, the evidence for this role is inconclusive. More direct evidence obtained by manipulating annexin II levels in cells is still required. We have attempted to do this by generating stably transfected PC12 cell lines expressing proteins which elevate or lower functional annexin II levels and using these cell lines to investigate Ca(2+)-dependent exocytosis. Three cell lines were generated: one expressing an annexin II mutant which aggregates annexin II in at least a proportion of the cells, thereby removing functional protein from the cell; a mixed clonal cell line constitutively overexpressing human annexin II; and a clonal cell line capable of over-expressing annexin II in the presence of sodium butyrate. After digitonin permeabilization, Ca(2+)-dependent dopamine release from these cell lines was compared with that from control nontransfected cells, and, in addition, release was compared in induced to uninduced cells. There were no significant differences in Ca(2+)-dependent exocytosis between any of the transfected cell lines before or after induction and the control cells. In addition, nontransfected PC12 cells treated with nerve growth factor, which elevates annexin II levels severalfold, failed to increase Ca(2+)-dependent exocytosis after digitonin permeabilization, compared with control cells. We conclude that annexin II is not an important regulator of Ca(2+)-dependent exocytosis in PC12 cells.     

The C2B domain of synaptotagmin I is a Ca2+-binding module

Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release. The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear. The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions. To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques. The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization. NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+). These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.     

Detergent solubilization of the endocytic Ca(2+)-independent hyaluronan receptor from rat liver endothelial cells and separation from a Ca(2+)-dependent hyaluronan-binding activity.

Rat liver sinusoidal endothelial cells (LECs) mediate the removal of hyaluronan (HA) from the circulation via a specific Ca(2+)-independent endocytic receptor. To characterize the receptor biochemically, detergent-soluble extracts were prepared from crude LEC membranes. Using a dot blot assay to quantitate 125I-HA binding activity in CHAPS-solubilized membranes, we detected not only specific Ca(2+)-independent but also specific Ca(2+)-dependent HA-binding activity. Both HA-binding activities behave as integral membrane-associated proteins; they are not released from LEC membranes by treatment at pH 11, and they require detergent for extraction. The Ca(2+)-independent HA receptor was inactivated by treatment at 56 degrees C for 30 min or with 200 mM DTT at 4 degrees C for 30 min, whereas the Ca(2+)-dependent activity actually increased by 75% after treatment at 56 degrees C and only 20% of the Ca(2+)-dependent activity was lost after DTT treatment. A two-cycle membrane extraction protocol using CHAPS partially separated the two HA-binding activities. Eight millimolar KCl and 0.5% CHAPS extracted approximately 50% of the Ca(2+)-independent HA receptor, but only 4-11% of the Ca(2+)-dependent activity. When the KCl and CHAPS concentrations were increased to 2.0 M and 1.5%, respectively, the remaining HA receptor, as well as 89-96% of the Ca(2+)-dependent activity, was then extracted. The Ca(2+)-independent and Ca(2+)-dependent activities could also be further separated using Sephacryl S-400 gel filtration chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake

Store-operated Ca(2+) channels, which are activated by the emptying of intracellular Ca(2+) stores, provide one major route for Ca(2+) influx. Under physiological conditions of weak intracellular Ca(2+) buffering, the ubiquitous Ca(2+) releasing messenger InsP(3) usually fails to activate any store-operated Ca(2+) entry unless mitochondria are maintained in an energized state. Mitochondria rapidly take up Ca(2+) that has been released by InsP(3), enabling stores to empty sufficiently for store-operated channels to activate. Here, we report a novel role for mitochondria in regulating store-operated channels under physiological conditions. Mitochondrial depolarization suppresses store-operated Ca(2+) influx independently of how stores are depleted. This role for mitochondria is unrelated to their actions on promoting InsP(3)-sensitive store depletion, can be distinguished from Ca(2+)-dependent inactivation of the store-operated channels and does not involve changes in intracellular ATP, oxidants, cytosolic acidification, nitric oxide or the permeability transition pore, but is suppressed when mitochondrial Ca(2+) uptake is impaired. Our results suggest that mitochondria may have a more fundamental role in regulating store-operated influx and raise the possibility of bidirectional Ca(2+)-dependent crosstalk between mitochondria and store-operated Ca(2+) channels.     

A helix-breaking mutation in the epithelial Ca(2+) channel TRPV5 leads to reduced Ca(2+)-dependent inactivation

TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca(2+) and is tightly regulated by intracellular Ca(2+) concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca(2+) concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca(2+)-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (τ) and half maximal inhibition constant (IC(50)) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca(2+) permeability. Out of different point mutations created to characterize the importance of M490 in Ca(2+)-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca(2+)-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca(2+)-dependent inactivation.     

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.