Functional specialization of presynaptic Cav2.3 Ca2+ channels

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.     

The Ca2+ antagonists binding cytosolic protein has properties of the Ca2+ channel.

In our previous work (Krizanová et al. 1989) we have described a protein from rabbit skeletal muscle cytosolic fraction, which is able to bind dihydropyridines and phenothiazines. In the present work conclusive evidence is provided for the ability of the phospholipid-reconstituted cytosolic protein to transport calcium. The calcium transport was stimulated by BAY K 8644 and inhibited in the presence of PN 200-110. Our observations were confirmed also by electrophysiological measurements on planar lipid bilayers. The possibility that the cytosolic fraction was contaminated with membranes could be definitely ruled out. Nevertheless, the nature of the protein under study is still in the frame of guess.     

The voltage-gated Ca2+ channel is the Ca2+ sensor of fast neurotransmitter release

Previously it demonstrated that in the absence of Ca²⁺ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca²⁺] _i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217–220.). These studies designate Ca²⁺ entry as opposed to [Ca²⁺] _i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca²⁺ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein–protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J.; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J. 15:4100–4110; Wiser, O., Trus, M., Hernandez, A., Renström, E., Barg, S., Rorsman, P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248–253). The kinetics of Ca_v1.2 (Lc-type) and Ca_v2.2 (N-type) Ca²⁺ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin I, a vesicle-associated protein, accelerated the activation kinetics indicating Ca_v2.2 coupling to the vesicle. The unique modifications of Ca_v1.2 and Ca_v2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Ca_v1.2 cytosolic domain Lc_753–893, acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca²⁺ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca²⁺-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca²⁺-dependent CAV (RRP) release, initiated by the binding of Ca²⁺ to the channel, upstream to intracellular Ca²⁺ sensor thus establishing the Ca²⁺ channel as the Ca²⁺ sensor of neurotransmitter release.     

The C-terminus of human Ca(v)2.3 voltage-gated calcium channel interacts with alternatively spliced calmodulin-2 expressed in two human cell lines

Ca(v)2.3 containing voltage-activated Ca(2+) channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca(2+) which can both, facilitate and inhibit the influx of Ca(2+) ions through Ca(v)2.3. The facilitated Ca(2+) influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca(2+) mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca(v)2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca(v)2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.     

电压门控钙通道钙依赖性易化和失活的分子机制

电压门控钙通道受钙依赖性易化和失活两种相互对立的反馈机制调节.不同浓度的钙离子,通过作为钙感受器的钙调蛋白的介导,主要与钙通道α1亚基羧基端的多个不连续片段发生复杂的相互作用,分别引发钙依赖性易化和失活.钙/钙调蛋白依赖性蛋白激酶Ⅱ及其它钙结合蛋白等也参与此调节过程.新近研究表明,钙通道的钙依赖性调节机制失衡与心律失常等的发病机制密切相关.     

The assembly and intermolecular properties of the hsp70-Hop-hsp90 molecular chaperone complex

The highly coordinated interactions of several molecular chaperones, including hsp70 and hsp90, are required for the folding and conformational regulation of a variety of proteins in eukaryotic cells, such as steroid hormone receptors and many other signal transduction regulators. The protein called Hop serves as an adaptor protein for hsp70 and hsp90 and is thought to optimize their functional cooperation. Here we characterize the assembly of the hsp70-Hop-hsp90 complex and reveal interactions that cause conformational changes between the proteins in the complex. We found that hsp40 plays an integral role in the assembly by enhancing the binding of hsp70 to the Hop complex. This is accomplished by stimulating the conversion of hsp70-ATP to hsp70-ADP, the hsp70 conformation favored for Hop binding. The hsp70-Hop-hsp90 complex is highly dynamic, as has been observed previously for hsp90 in its interaction with client proteins. Nonetheless, hsp90 binds with high affinity to Hop (K(d) = 90 nm), and this binding is not affected by hsp70. hsp70 binds with lower affinity to Hop (K(d) = 1.3 microm) on its own, but this affinity is increased (K(d) = 250 nm) in the presence of hsp90. hsp90 also reduces the number of hsp70 binding sites on the Hop dimer from two sites in the absence of hsp90 to one site in its presence. Hop can inhibit the ATP binding and p23 binding activity of hsp90, yet this can be reversed if hsp70 is present in the complex. Taken together, our results suggest that the assembly of hsp70-Hop-hsp90 complexes is selective and influences the conformational state of each protein.     

Localization of the activation gate of a voltage-gated Ca2+ channel

Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K+ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca2+ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the alpha1 subunit of P/Q type Ca2+ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.     

The L-type voltage-gated Ca2+ channel is the Ca2+ sensor protein of stimulus-secretion coupling in pancreatic beta cells

L-type voltage-gated Ca2+ channels (Cav1.2) mediate a major part of insulin secretion from pancreatic beta-cells. Cav1.2, like other voltage-gated Ca2+ channels, is functionally and physically coupled to synaptic proteins. The tight temporal coupling between channel activation and secretion leads to the prediction that rearrangements within the channel can be directly transmitted to the synaptic proteins, subsequently triggering release. La3+, which binds to the polyglutamate motif (EEEE) comprising the selectivity filter, is excluded from entry into the cells and has been previously shown to support depolarization-evoked catecholamine release from chromaffin and PC12 cells. Hence, voltage-dependent trigger of release relies on Ca2+ ions bound at the EEEE motif and not on cytosolic Ca2+ elevation. We show that glucose-induced insulin release in rat pancreatic islets and ATP release in INS-1E cells are supported by La3+ in nominally Ca2+-free solution. The release is inhibited by nifedipine. Fura 2 imaging of dispersed islet cells exposed to high glucose and La3+ in Ca2+-free solution detected no change in fluorescence; thus, La3+ is excluded from entry, and Ca2+ is not significantly released from intracellular stores. La3+ by interacting extracellularlly with the EEEE motif is sufficient to support glucose-induced insulin secretion. Voltage-driven conformational changes that engage the ion/EEEE interface are relayed to the exocytotic machinery prior to ion influx, allowing for a fast and tightly regulated process of release. These results confirm that the Ca2+ channel is a constituent of the exocytotic complex [Wiser et al. (1999) PNAS 96, 248-253] and the putative Ca2+-sensor protein of release.     

Switch of voltage-gated K+ channel expression in the plasma membrane of chondrogenic cells affects cytosolic Ca2+-oscillations and cartilage formation

Background

Understanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stem cells in cartilage tissue engineering, which is a promising approach of regenerative therapy of joint diseases. Ion channels, membrane potential and Ca²⁺-signaling are important regulators of cell proliferation and differentiation. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and ultimately cartilage formation.

Methodology/Principal Findings

Using patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated Na_V1.4, K_V1.1, K_V1.3 and K_V4.1 channels, although K_V1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of Na_V1.4 channels, had no effect on cartilage formation. In contrast, presence of 20 mM of the K⁺ channel blocker tetraethyl-ammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced K_V currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV). High-frequency Ca²⁺-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive K_V1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive K_V4.1 did not affect cartilage formation.

Conclusions/Significance

These data demonstrate that the differentiation and proliferation of chondrogenic cells depend on rapid Ca²⁺-oscillations, which are modulated by K_V-driven membrane potential changes. K_V1.1 function seems especially critical during the final commitment period. We show the critical role of voltage-gated cation channels in the differentiation of non-excitable cells with potential therapeutic use.     

Functional architecture of the inner pore of a voltage-gated Ca2+ channel

The inner pore of voltage-gated Ca2+ channels (VGCCs) is functionally important, but little is known about the architecture of this region. In K+ channels, this part of the pore is formed by the S6/M2 transmembrane segments from four symmetrically arranged subunits. The Ca2+ channel pore, however, is formed by four asymmetric domains of the same (alpha1) subunit. Here we investigated the architecture of the inner pore of P/Q-type Ca2+ channels using the substituted-cysteine accessibility method. Many positions in the S6 segments of all four repeats of the alpha1 subunit (Ca(v)2.1) were modified by internal methanethiosulfonate ethyltrimethylammonium (MTSET). However, the pattern of modification does not fit any known sequence alignment with K+ channels. In IIS6, five consecutive positions showed clear modification, suggesting a likely aqueous crevice and a loose packing between S6 and S5 segments, a notion further supported by the observation that some S5 positions were also accessible to internal MTSET. These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels. Interestingly some residues in IIIS6 and IVS6 whose mutations in L-type Ca2+ channels affect the binding of dihydropyridines and phenylalkylamines and are thought to face the pore appeared not to react with internal MTSET. Probing with qBBr, a rigid thiol-reactive agent with a dimension of 12 angstroms x 10 angstroms x 6 angstroms suggests that the inner pore can open to >10 angstroms. This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.     

Side chain orientation in the selectivity filter of a voltage-gated Ca2+ channel

Four glutamate residues (EEEE locus) are essential for ion selectivity in voltage-gated Ca(2+) channels, with ion-specific differences in binding to the locus providing the basis of selectivity. Whether side chain carboxylates or alternatively main chain carbonyls of these glutamates project into the pore to form the ion-binding locus has been uncertain. We have addressed this question by examining effects of sulfhydryl-modifying agents (methanethiosulfonates) on 20 cysteine-substituted mutant forms of an L-type Ca(2+) channel. Sulfhydryl modifiers partially blocked whole oocyte Ba(2+) currents carried by wild type channels, but this block was largely reversed with washout. In contrast, each of the four EEEE locus glutamate --> cysteine mutants (0 position) was persistently blocked by sulfhydryl modifiers, indicating covalent attachment of a modifying group to the side chain of the substituted cysteine. Cysteine substitutions at positions immediately adjacent to the EEEE locus glutamates (+/-1 positions) were also generally susceptible to sulfhydryl modification. Sulfhydryl modifiers had lesser effects on channels substituted one position further from the EEEE locus (+/-2 positions). These results indicate that the carboxylate-bearing side chains of the EEEE locus glutamates and their immediate neighbors project into the water-filled lumen of the pore to form an ion-binding locus. Thus the structure of the Ca(2+) channel selectivity filter differs substantially from that of ancestral K(+) channels.     

Downstream caspases are novel targets for the antiapoptotic activity of the molecular chaperone hsp70

The response of cancer cells to apoptosis-inducing agents can be characterized by 2 opposing factors, the proapoptotic caspase cascade and the antiapoptotic stress protein Hsp70. We show here that these factors interact in U-937 leukemia cells induced to apoptosis with anticancer drugs, etoposide and adriamycin (ADR). The protective effect of Hsp70 was verified using 2 approaches: mild heat stress and transfection-mediated overexpression of the Hsp70 gene. The increase in Hsp70 levels attained by these 2 methods was found to postpone caspase activation for 12-18 hours. An in vitro assay was developed using mouse myeloma NS0/1 cells, which lack the expression of Hsp70. Measurement of DEVD-ase activity in extracts of apoptotic NS0/1 cells incubated with purified Hsp70 showed that Hsp70 reduced caspase activity by up to 50% of its control value in a dose-dependent manner. The hypothesis that the inhibitory effect of Hsp70 on caspase-3/7 activity related to a direct interaction between Hsp70 and the caspases was tested by reciprocal immunoprecipitations and Far-western analyses. These tests were performed with extracts of Hsp70-overexpressing, control, and ADR-treated U-937 cells and using anti-caspase-3, caspase-7, and anti-Hsp70 antibodies, and the data clearly showed that Hsp70 was able to interact with the proforms of these caspases in cell lysates and with reconstituted purified proteins but did not bind the activated forms of either caspase-3 or -7. This association was also corroborated by a novel, enzyme-linked immunosorbent assay-like assay, protein interaction assay, that combined the advantages of immunoprecipitation and immunoblotting in a 96-well microplate-based assay. Thus, Hsp70 may act to suppress caspase-dependent apoptotic signaling through binding the precursor forms of both caspase-3 and caspase-7 and preventing their maturation.     

The Zn2+-transporting pathways in pancreatic beta-cells: a role for the L-type voltage-gated Ca2+ channel

In pancreatic beta-cells Zn(2+) is crucial for insulin biosynthesis and exocytosis. Despite this, little is known about mechanisms of Zn(2+) transport into beta-cells or the regulation and compartmentalization of Zn(2+) within this cell type. Evidence suggests that Zn(2+) in part enters neurons and myocytes through specific voltage-gated calcium channels (VGCC). Using a Zn(2+)-selective fluorescent dye with high affinity and quantum yield, FluoZin-3 AM and the plasma membrane potential dye DiBAC(4)(3) we applied fluorescent microscopy techniques for analysis of Zn(2+)-accumulating pathways in mouse islets, dispersed islet cells, and beta-cell lines (MIN6 and beta-TC6f7 cells). Because the stimulation of insulin secretion is associated with cell depolarization, Zn(2+) (5-10 mum) uptake was analyzed under basal (1 mm glucose) and stimulatory (10-20 mm glucose, tolbutamide, tetraethylammonium, and high K(+)) conditions. Under both basal and depolarized states, beta-cells were capable of Zn(2+) uptake, and switching from basal to depolarizing conditions resulted in a marked increase in the rate of Zn(2+) accumulation. Importantly, L-type VGCC (L-VGCC) blockers (verapamil, nitrendipine, and nifedipine) as well as nonspecific inhibitors of Ca(2+) channels, Gd(3+) and La(3+), inhibited Zn(2+) uptake in beta-cells under stimulatory conditions with little or no change in Zn(2+) accumulation under low glucose conditions. To determine the mechanism of VGCC-independent Zn(2+) uptake the expression of a number of ZIP family Zn(2+) transporter mRNAs in islets and beta-cells was investigated. In conclusion, we demonstrate for the first time that, in part, Zn(2+) transport into beta-cells takes place through the L-VGCC. Our investigation demonstrates direct Zn(2+) accumulation in insulin-secreting cells by two pathways and suggests that the rate of Zn(2+) transport across the plasma membrane is dependent upon the metabolic status of the cell.     

Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger

Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na+/Ca2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the first five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using glutathione S-transferase (GST) pull-down assay, we demonstrated that the intracellular loop, but not the N- or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na+/Ca2+ exchangers consisting of N- or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. Although expression of N-terminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N- and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Co-immunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.     

Histidine residues in the IS3-IS4 loop are critical for nickel-sensitive inhibition of the Cav2.3 calcium channel

We recently reported that a histidine (H191) in the S3-S4 loop of domain I is critical for nickel inhibition of the Cav3.2 T-type Ca2+ channel. As in Cav3.2, two histidine residues are commonly found in the IS3-IS4 loops of mammalian Cav2.3 Ca2+ channels, which are also blocked by low micromolar concentrations of nickel. We show here by site-directed mutagenesis and electrophysiology that both residues contribute to the nickel sensitivity of Cav2.3, with H183 being more critical than H179. These findings strongly suggest that both H179 and H183 in the IS3-IS4 loop are essential structural determinants required for nickel sensitive inhibition of the Cav2.3.     

The N-terminal portion of the main cytosolic loop mediates K+ sensitivity in the retinal rod Na+/Ca2+-K+-exchanger.

Two types of Na+/Ca2+-exchangers have been characterized in the literature: The first is the cardiac, skeletal muscle and brain type, which exchanges 1 Ca2+ for 3 Na+, the second, found in retinal photosensor cells, transports 1 Ca2+ and 1 K+ in exchange for 4 Na+. The present work describes the properties of chimeric constructs of the two exchanger types. Ca2+ gel overlay experiments have identified a high affinity (Kd in the 1 microM range) Ca2+-binding domain between Glu601 and Asp733 in the main cytosolic loop of the retinal protein, just after transmembrane domain 5. Insertion of the retinal Ca2+-binding domain in the cytosolic loop of the cardiac exchanger conferred K+-dependence to the Ca2+ uptake activity of the chimeric constructs expressed in HeLa cells. The apparent Km of the K+ effect was about 1 mM. Experiments with C-terminally truncated versions of the retinal insert indicated that the sequence between Leu643 and Asp733 was critical in mediating K+ sensitivity of the recombinant chimeras. Thus, the high affinity Ca2+-binding domain in the main cytosolic loop of the retinal exchanger may regulate the activity of the retinal protein by binding Ca2+, and by conferring to it K+ sensitivity.     

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.