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• W1971742114 abstract "Neuronal Ca2+sensor protein-1 (NCS-1) is a member of the Ca2+ binding protein family, with three functional Ca2+ binding EF-hands and an N-terminal myristoylation site. NCS-1 is expressed in brain and heart during embryonic and postnatal development. In neurons, NCS-1 facilitates neurotransmitter release, but both inhibition and facilitation of the Ca2+ current amplitude have been reported. In heart, NCS-1 co-immunoprecipitates with K+channels and modulates their activity, but the potential effects of NCS-1 on cardiac Ca2+ channels have not been investigated. To directly assess the effect of NCS-1 on the various types of Ca2+ channels we have co-expressed NCS-1 inXenopus oocytes, with CaV1.2, CaV2.1, and CaV2.2 Ca2+ channels, using various subunit combinations. The major effect of NCS-1 was to decrease Ca2+ current amplitude, recorded with the three different types of α1 subunit. When expressed with CaV2.1, the depression of Ca2+ current amplitude induced by NCS-1 was dependent upon the identity of the β subunit expressed, with no block recorded without β subunit or with the β3 subunit. Current-voltage and inactivation curves were also slightly modified and displayed a different specificity toward the β subunits. Taken together, these data suggest that NCS-1 is able to modulate cardiac and neuronal voltage-gated Ca2+ channels in a β subunit specific manner. Neuronal Ca2+sensor protein-1 (NCS-1) is a member of the Ca2+ binding protein family, with three functional Ca2+ binding EF-hands and an N-terminal myristoylation site. NCS-1 is expressed in brain and heart during embryonic and postnatal development. In neurons, NCS-1 facilitates neurotransmitter release, but both inhibition and facilitation of the Ca2+ current amplitude have been reported. In heart, NCS-1 co-immunoprecipitates with K+channels and modulates their activity, but the potential effects of NCS-1 on cardiac Ca2+ channels have not been investigated. To directly assess the effect of NCS-1 on the various types of Ca2+ channels we have co-expressed NCS-1 inXenopus oocytes, with CaV1.2, CaV2.1, and CaV2.2 Ca2+ channels, using various subunit combinations. The major effect of NCS-1 was to decrease Ca2+ current amplitude, recorded with the three different types of α1 subunit. When expressed with CaV2.1, the depression of Ca2+ current amplitude induced by NCS-1 was dependent upon the identity of the β subunit expressed, with no block recorded without β subunit or with the β3 subunit. Current-voltage and inactivation curves were also slightly modified and displayed a different specificity toward the β subunits. Taken together, these data suggest that NCS-1 is able to modulate cardiac and neuronal voltage-gated Ca2+ channels in a β subunit specific manner. neuronal Ca2+ sensor protein-1 Ca2+ entry through voltage-gated Ca2+ channels is essential for various cellular processes that include muscle contraction, pacemaker activity, synaptic transmission, or gene expression. Several types of Ca2+channels have been characterized (T, L, N, P/Q, and R) that appear to play a specific role in each of these functions. These channels share a common architecture composed of a major α1 subunit (for which ten genes are known) tightly associated with regulatory subunits α2-δ (four different genes), β (four genes), and possibly γ (eight genes) in a functional multimeric complex (1Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1957) Google Scholar, 2Hanlon M.R. Wallace B.A. Biochemistry. 2002; 41: 2886-2894Crossref PubMed Scopus (88) Google Scholar, 3Hofmann F. Lacinova L. Klugbauer N. Rev. Physiol Biochem. Pharmacol. 1999; 139: 33-87Crossref PubMed Google Scholar, 4Birnbaumer L. Qin N. Olcese R. Tareilus E. Platano D. Costantin J. Stefani E. J. Bioenerg. Biomembr. 1998; 30: 357-375Crossref PubMed Scopus (202) Google Scholar). This molecular diversity, further expanded by the existence of several splice variants for each of these genes, produces a large number of possible Ca2+ channel subunit combinations with different pharmacological and biophysical properties and specific cellular and subcellular localization (5Felix R. Receptors. Channels. 1999; 6: 351-362PubMed Google Scholar). The precise regulation of the Ca2+ influx in response to various physiological situations is further controlled by several regulatory mechanisms, working at different levels, including channel expression, localization, or activity, via additional interactions with modulatory proteins. Several Ca2+-dependent feedback mechanisms sense incoming Ca2+ ions to finely tune channel activity to the cellular Ca2+ demands and prevent cytotoxic Ca2+ overload (6Budde T. Meuth S. Pape H.C. Nat. Rev. Neurosci. 2002; 3: 873-883Crossref PubMed Scopus (169) Google Scholar). These mechanisms use Ca2+-sensing proteins and are specific of a given type of Ca2+ channel. It has been shown, for example, that Ca2+-dependent inactivation of the L-type Ca2+ channel (encoded by the CaV1.2 α1 subunit) is governed by a Ca2+-driven interaction between calmodulin and the C-terminal tail of the channel α1 subunit (7Peterson B.Z. Lee J.S. Mulle J.G. Wang Y. de Leon M. Yue D.T. Biophys. J. 2000; 78: 1906-1920Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 8Romanin C. Gamsjaeger R. Kahr H. Schaufler D. Carlson O. Abernethy D.R. Soldatov N.M. FEBS Lett. 2000; 487: 301-306Crossref PubMed Scopus (67) Google Scholar, 9Peterson B.Z. DeMaria C.D. Adelman J.P. Yue D.T. Neuron. 1999; 22: 549-558Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar, 10Zuhlke R.D. Pitt G.S. Deisseroth K. Tsien R.W. Reuter H. Nature. 1999; 399: 159-162Crossref PubMed Scopus (743) Google Scholar). A similar functional interaction also appears to exist on the P/Q-type Ca2+ channel (encoded by the CaV2.1 subunit), the major channel type involved in synaptic transmission in the mammalian central nervous system (11Lee A. Westenbroek R.E. Haeseleer F. Palczewski K. Scheuer T. Catterall W.A. Nat. Neurosci. 2002; 5: 210-217Crossref PubMed Scopus (165) Google Scholar, 12DeMaria C.D. Soong T.W. Alseikhan B.A. Alvania R.S. Yue D.T. Nature. 2001; 411: 484-489Crossref PubMed Scopus (341) Google Scholar, 13Lee A. Wong S.T. Gallagher D. Li B. Storm D.R. Scheuer T. Catterall W.A. Nature. 1999; 399: 155-159Crossref PubMed Scopus (1004) Google Scholar). The neuronal Ca2+ sensor protein-1 (NCS-1),1 the mammalian homologue of frequenin, belongs to a group of small Ca2+-binding proteins comprising four EF-hand motifs, three of which are able to bind Ca2+ in the micromolar (EF-2) or submicromolar (EF-3,4) range (14Olafsson P. Wang T. Lu B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8001-8005Crossref PubMed Scopus (87) Google Scholar, 15Bourne Y. Dannenberg J. Pollmann V. Marchot P. Pongs O. J. Biol. Chem. 2001; 276: 11949-11955Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 16Burgoyne R.D. Weiss J.L. Biochem. J. 2001; 353: 1-12Crossref PubMed Scopus (379) Google Scholar). NCS-1 also contains an N-terminal myristoylation site (17McFerran B.W. Weiss J.L. Burgoyne R.D. J. Biol. Chem. 1999; 274: 30258-30265Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). NCS-1 has been shown to facilitate synapse formation, spontaneous and/or evoked neurotransmitter release, paired-pulse facilitation, and exocytosis in several cell types (18Tsujimoto T. Jeromin A. Saitoh N. Roder J.C. Takahashi T. Science. 2002; 295: 2276-2279Crossref PubMed Scopus (184) Google Scholar, 19Weiss J.L. Archer D.A. Burgoyne R.D. J. Biol. Chem. 2000; 275: 40082-40087Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 20Wang C.Y. Yang F. He X. Chow A. Du J. Russell J.T. Lu B. Neuron. 2001; 32: 99-112Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 21McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 22Chen X.L. Zhong Z.G. Yokoyama S. Bark C. Meister B. Berggren P.O. Roder J. Higashida H. Jeromin A. J. Physiol. 2001; 532: 649-659Crossref PubMed Scopus (47) Google Scholar). NCS-1 interacts directly with phosphatidylinositol 4-kinase in yeast (23Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (220) Google Scholar), COS-7 (24Zhao X. Varnai P. Tuymetova G. Balla A. Toth Z.E. Oker-Blom C. Roder J. Jeromin A. Balla T. J. Biol. Chem. 2001; 276: 40183-40189Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and chromaffin cells (25Pan C.Y. Jeromin A. Lundstrom K. Yoo S.H. Roder J. Fox A.P. J. Neurosci. 2002; 22: 2427-2433Crossref PubMed Google Scholar), leading to the hypothesis that part of the effects mediated by NCS-1 could involve modifications in cellular trafficking through regulation of the phosphatidylinositol signaling pathway, thereby affecting vesicular transport and recycling, as well as inositol 1,4,5-trisphosphate-sensitive Ca2+ stores (24Zhao X. Varnai P. Tuymetova G. Balla A. Toth Z.E. Oker-Blom C. Roder J. Jeromin A. Balla T. J. Biol. Chem. 2001; 276: 40183-40189Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 25Pan C.Y. Jeromin A. Lundstrom K. Yoo S.H. Roder J. Fox A.P. J. Neurosci. 2002; 22: 2427-2433Crossref PubMed Google Scholar). However, a possible involvement of NCS-1 in expression and regulation of voltage-gated Ca2+ channels has also been proposed. Overexpression of a dominant-negative mutant of NCS-1, which displays impaired Ca2+-dependent conformational changes (19Weiss J.L. Archer D.A. Burgoyne R.D. J. Biol. Chem. 2000; 275: 40082-40087Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), or direct loading of presynaptic nerve terminals with NCS-1 suggested that voltage-independent inhibition, as well as activity-dependent facilitation of P/Q-type Ca2+ channels (CaV2.1), could be controlled by NCS-1, possibly via direct protein-protein interactions (18Tsujimoto T. Jeromin A. Saitoh N. Roder J.C. Takahashi T. Science. 2002; 295: 2276-2279Crossref PubMed Scopus (184) Google Scholar). Effects on N-type Ca2+ channel (CaV2.2) properties have also been reported (20Wang C.Y. Yang F. He X. Chow A. Du J. Russell J.T. Lu B. Neuron. 2001; 32: 99-112Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and the expression of NCS-1 in mammalian cardiac myocytes and subsequent effect on K+ channel expression (26Guo W. Malin S.A. Johns D.C. Jeromin A. Nerbonne J.M. J. Biol. Chem. 2002; 277: 26436-26443Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) gave rise to the possibility that NCS-1 may regulate multiple types of Ca2+ channels and other voltage-dependent ion channels, not only in neurons (27Weiss J.L. Burgoyne R.D. Trends Neurosci. 2002; 25: 489-491Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In a first step to explore this possibility, we have co-expressed NCS-1 with three different types of Ca2+ channel, CaV1.2, CaV2.1, and CaV2.2, associated with different combinations of auxiliary β subunit, and measured the resulting Ba2+ and Ca2+ currents. These combinations are likely to be expressed in different cell types where they represent potential targets for NCS-1 effects. Our goal was to explore the effect of NCS-1 on both Ca2+ channel expression and properties and to provide a first description of the molecular requirements necessary for NCS-1 effects on Ca2+channel that may help in the understanding of the precise mode of action of this Ca2+-binding protein. We have, however, focused this study on the P/Q-type (CaV2.1) Ca2+ channels, which seem to be a primary target in various cell types (28Weiss J.L. Burgoyne R.D. J. Biol. Chem. 2001; 276: 44804-44811Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Our results show that NCS-1 down-regulates expression of L-, N-, and P/Q-type Ca2+ channels in a β subunit-specific manner and induces minor modifications of the electrophysiological properties of the channel. We provide evidence of direct functional effects of NCS-1, in addition to modifications in the expression level and/or trafficking of the channels to the membrane. The following cDNA were used, and the GenBankTM accession number is provided: CaV1.2 (α1C), M67515; CaV2.1 (α1A), M64373; CaV2.2 (α1B),D14157; NCS-1, L27421; β1b, X61394; β2a,M80545; β3, M88751; β4, L02315; and α2-δ1, M86621. Mutations NCS-1E120Q and NCS-1G2A have been described previously (29O'Callaghan D.W. Ivings L. Weiss J.L. Ashby M.C. Tepikin A.V. Burgoyne R.D. J. Biol. Chem. 2002; 277: 14227-14237Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Ca2+ channel subunits were subcloned into the pmt2 vector, whereas NCS-1 and its mutants were subcloned into pcDNA3 (Invitrogen). Xenopus laevis oocyte preparation and injection were performed as described previously (30Cens T. Mangoni M.E. Richard S. Nargeot J. Charnet P. Pflugers Arch. 1996; 431: 771-774PubMed Google Scholar). Each oocyte was injected with 5–10 nl of a cDNA mixture containing the α1+α2δ+β+NCS-1 cDNAs at ∼0.3 ng/nl with a ratio of 1:2:3:1. When one or more of these cDNAs was omitted, cDNA concentrations were kept constant by addition of the appropriate volume of deionized water. Oocytes were kept for 2 to 4 days before recordings at 18 °C and under gentle agitation. Whole-cell Ba2+ currents were recorded under two-electrode voltage clamp using the GeneClamp 500 amplifier (Axon Instruments, Union City, CA). Current and voltage electrodes (less than 1 megohm) were filled with 3 m KCl, pH 7.2, with KOH. Ba2+ and Ca2+ current recordings were performed after injection of BAPTA (∼50 nl of the following (in mm): BAPTA-free acid (Sigma), 100; CsOH, 10; HEPES, 10; pH 7.2 with CsOH) using the following bathing solution (in mm): BaOH/CaOH, 10; TEAOH, 20; NMDG, 50; CsOH, 2; HEPES, 10; pH 7.2, with methanesulfonic acid. Currents were filtered and digitized using a DMA-Tecmar Labmaster and subsequently stored on a Pentium-based personal computer using the pClamp software (version 6.02; Axon Instruments). Ba2+ or Ca2+currents were recorded during a 400-ms test pulse from −80 to +10 mV. Current amplitudes were measured at the peak of the current. Comparisons of averaged amplitudes between batches were always made with amplitudes measured the same day after injection. Comparisons between similar experiments were made by normalizing all averaged amplitudes with respect to the control current amplitude set as 100%. Isochronal steady-state inactivation curves (2.5 s of conditioning voltage followed by a 400-ms test pulse to +10 mV) were fitted using the equation, I/I max =R in + (1 − R in)/(1 + exp((V − V in)/k)), where I is the current amplitude measured during the test pulse at +10 mV for conditioning voltages varying from −80 to +50 mV,I max is the current amplitude measured during the test pulse for a conditioning voltage to −80 mV,V in is the potential for half-inactivation,V is the voltage, k is the slope factor, andR in is the proportion of non-inactivating current. Current to voltage curves were fitted using the equation,I/I max =G*(V − E rev)/(1 + exp((V − V act)/k)), where I is the current amplitude measured during voltage steps varying from −80 to +50 mV, I max is the peak current amplitude measured at the minimum of the current-voltage curve, G is the normalized macroscopic conductance,E rev is the apparent reversal potential,V act is the potential for half-activation,V is the value of the voltage step, and k is a slope factor. Inactivation kinetics were estimated by fitting Ba2+current decay with two exponential components using the equation,I(t) =A 1e(−t/τ1) +A 2e(−t/τ2) + C, whereI is the current amplitude, t is the time, τ1, τ2, A 1, andA 2 represent the time constants and amplitudes of the two components, and C is a constant. The proportion of the slow time constant (%τ2) is the ratioA 2/(A 1+A 2). Several independent experiments (N, number of batches of oocytes injected) were performed, always including a control group without NCS-1 expressed. These experiments always gave the same statistical result, and the total number of recordings (fromn oocytes of these N experiments) are thus presented. All values are presented as mean ± S.E., and comparison between groups of oocytes were evaluated using a Student'st test, with a statistical significance set at thep value <0.05. Selected oocytes were first homogenized in the lysis buffer (5 μl/oocyte of 20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 50 mm NaF, 10 mmβ-glycerophosphate, and 5 mmNa4P2O7) and centrifuged at 4 °C and 14000 rpm for 5 min. The upper aqueous phase was collected and subjected to 10% (see Fig. 2 C) or 15% SDS-polyacrylamide gel electrophoresis (loaded with ∼3 oocytes/lane). Proteins were electrotransferred to nitrocellulose filters. For immunological detection, the blots were first blocked for 1 h with 8% non-fat milk powder in TBST (10 mm Tris-HCl, pH 8, 150 mm NaCl, 0.1% Tween 20), incubated overnight at 4 °C with anti-NCS-1 (Zymol at 1/1000 dilution in 0.1% bovine serum albumin in TBST) or anti-CaV2.1 antibodies (Alomone Laboratories Jerusalem, Israel), and after five washes, incubated with an anti-rabbit antibody coupled to horseradish peroxidase. Antibody binding was detected by chemiluminescence (PerkinElmer Life Sciences; 1/10000 in 0.1% bovine serum albumin, 0.8% nonfat milk powder in TBST). Correct expression of NCS-1 was always checked after electrophysiological recordings. Previous works on chromaffin, human embryonic kidney 293, and COS-7 cells have shown that NCS-1 was endogenously expressed at non-negligible levels (24Zhao X. Varnai P. Tuymetova G. Balla A. Toth Z.E. Oker-Blom C. Roder J. Jeromin A. Balla T. J. Biol. Chem. 2001; 276: 40183-40189Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar,28Weiss J.L. Burgoyne R.D. J. Biol. Chem. 2001; 276: 44804-44811Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 2A. Jeromin, unpublished observations. In non-injectedX. laevis oocytes, the level of expression of the endogenous NCS-1 was barely detectable in Western-blots and much lower than in human embryonic kidney 293 and COS-7 cells (see Fig. 1 A). Thus X. laevis oocytes are a system of choice to study the functional effect of NCS-1 on voltage-gated Ca2+channels. In these oocytes, injection of the cDNA coding for rat NCS-1 led, as expected, to a massive expression of a protein of a molecular mass of ∼23 kDa, in accordance with the theoretical molecular mass of NCS-1 (21.9 kDa; see Fig. 1 B). In the following experiments, the oocytes used for current recordings were collected after recordings and submitted to a similar Western blot analysis to ensure that the NCS-1 protein was properly expressed. In a first set of experiments, the effects of NCS-1 were tested on Ba2+currents flowing through L-, N-, and P/Q-type Ca2+channels. For each Ca2+ channel type, this was done by co-injecting a mixture of cDNA containing β2, α2-δ, and the appropriate α1Ca2+ channel subunits (CaV1.2, CaV2.2, or CaV2.1, respectively) with either NCS-1 cDNA or water into two different batches of oocytes. After 2 to 4 days of incubation, Ba2+ current amplitudes were recorded from the two batches of oocytes injected the same day, during a single 400-ms-long depolarizing step to +10 mV from a holding potential of −80 mV and compared. Under these conditions, a clear decrease in the averaged Ba2+ current amplitude was seen upon co-expression of NCS-1 (see Fig.2 A). This effect was most pronounced in oocytes expressing the CaV2.2 Ca2+ channel, where the averaged Ba2+ current amplitude recorded in the batch of oocytes co-injected with rat NCS-1 cDNA (N = 1 experiment, n = 38 oocytes) was only 10% of the control current amplitude recorded from oocytes co-injected with H2O instead of the NCS-1 cDNA (N = 1, n = 35). However, a similar effect was also found when NCS-1 was co-injected with CaV1.2 (N = 3, n = 71; 52% of the control amplitude n = 70) or CaV2.1 Ca2+ channel subunits (N = 7,n = 206; 46% of the control amplituden = 162). The Ca2+ channel β subunit is an important determinant of the final Ca2+ current amplitude observed at the oocyte surface membrane and regulates many of its electrophysiological properties (4Birnbaumer L. Qin N. Olcese R. Tareilus E. Platano D. Costantin J. Stefani E. J. Bioenerg. Biomembr. 1998; 30: 357-375Crossref PubMed Scopus (202) Google Scholar, 31Perez-Reyes E. Castellano A. Kim H.S. Bertrand P. Baggstrom E. Lacerda A.E. Wei X.Y. Birnbaumer L. J. Biol. Chem. 1992; 267: 1792-1797Abstract Full Text PDF PubMed Google Scholar, 32Singer D. Biel M. Lotan I. Flockerzi V. Hofmann F. Dascal N. Science. 1991; 253: 1553-1557Crossref PubMed Scopus (441) Google Scholar). Hence, we tested the role of the β subunit on the effects of NCS-1. Using the same experimental approach, we co-expressed NCS-1 with the CaV2.1 Ca2+ channel either with the α2-δ subunit alone or with the α2-δ and one of the four β subunits (β1-β4). Again, in each case, the resulting current amplitude was compared with the current amplitude recorded in oocytes injected with the same combination of Ca2+ channel subunits but without NCS-1. Interestingly, when NCS-1 was co-expressed with CaV2.1 without β subunit, almost no effect on current amplitude was observed (N = 2,n = 32 and 26 for control). A similar result was also found upon co-expression of the β3 subunit (N = 12, n = 198, with controln = 198), whereas co-expression of NCS-1 with the CaV2.1 and β1, β2, or β4 subunit decreased the expressed Ba2+current amplitude to between 25 and 45% of their respective control values (see Fig. 2 B, N = 3, 7, 2 andn = 100, 206, 71 and n = 65, 162, 84 for controls, respectively). The lack of effect of NCS-1 on the CaV2.1 Ca2+ channel subunit, expressed alone or with the β3 subunit, could not be attributed to a deficit of NCS-1 protein in these oocytes, because a robust expression of NCS-1 was also detected in these oocytes by Western blot (seebottom of Fig. 2 B). On the other hand, to ensure that the decrease of the Ba2+ current amplitude obtained upon co-expression of NCS-1 with the β1, β2, or β4 subunit was not because of a deleterious effect on the expression of any cloned protein, we analyzed the level of expression of the CaV2.1 protein in these conditions by Western blot. Fig. 2 C demonstrates that a clear band of an approximate molecular mass of 250 kDa, matching of the theoretical molecular mass of the CaV2.1 subunit (251 kDa), could be specifically detected only in oocytes injected with the CaV2.1 subunit cDNA (+α2-δ+β2 subunits; see Fig.2 C, left lane). In oocytes co-injected with NCS-1 cDNA and the same combinations of Ca2+ channel subunits, this CaV2.1 immunoreactive band was also present at similar intensity (Fig. 2 C, middle lane, eachlane approximately loaded with three oocytes), whereas it was absent in non-injected oocytes (Fig. 2 C, right lane). These results therefore revealed a true, β subunit-specific effect of NCS-1 on the Ba2+ current amplitude that cannot be attributed to nonspecific down-regulation of protein expression. We next investigated whether co-expression of NCS-1 with the CaV2.1 subunit might have other functional consequences on Ca2+channel properties. Current-voltage curves and isochronal inactivation curves were constructed from currents recorded in oocytes expressing the CaV2.1 subunit with the α2-δ alone or with the β1, β2, or β3subunits, in the presence or absence of NCS-1. These recordings were performed using either Ba2+ or Ca2+ ions as extracellular permeant cations to track any Ca2+-specific modulation. As presented in Table I, in the presence of Ca2+ ions and in the absence of β subunit (α1A+α2-δ subunits), no modifications in the activation and inactivation parameters were observed upon co-expression of NCS-1. This lack of effect was also noted upon co-expression of the β1 or β3subunit (see Fig. 3 and Table I). Interestingly, when co-expressed with CaV2.1 and the β2a subunit, NCS-1 significantly depolarized the current-voltage curve (V act = −5.0 and −0.8 mV without and with NCS-1 respectively, p < 0.05) and reduced inactivation (R in = 69 and 56% respectively, p < 0.05). Similar effects were also found in the presence of extracellular Ba2+ (TableII) and thus were not Ca2+-dependent.Table IEffects of NCS-1 on Cav2.1 Ca2+ currentsCa2+currentsV actK actV inK inR inmV%α1A + α2 − δ (n = 3)17.1 ± 5.0−8.2 ± 1.7−7.1 ± 1.39.5 ± 2.00 ± 14α1A + α2 − δ + NCS-1 (n = 4)20.5 ± 5.4−13.3 ± 2.5−4.3 ± 1.710.7 ± 1.70 ± 3.5α1A + α2 − δ + β1 (n = 8)0.6 ± 1.1−4.6 ± 0.2−18.3 ± 0.45.5 ± 0.210 ± 1.0α1A + α2 − δ + β1 + NCS-1 (n = 8)−0.5 ± 1.0−4.9 ± 0.5−17.3 ± 0.75.7 ± 0.315 ± 3.2α1A + α2 − δ + β2 (n = 8)−5.1 ± 1.1−3.6 ± 0.5−6.1 ± 0.54.3 ± 1.169 ± 2.9α1A + α2 − δ + β2 + NCS-1 (n = 10)−0.8 ± 1.0aStatistically different from control without NCS-1 expressed (0.05 level).−4.1 ± 0.7−3.8 ± 1.35.5 ± 0.856 ± 3.9aStatistically different from control without NCS-1 expressed (0.05 level).α1A + α2 − δ + β3 (n = 7)1.3 ± 1.0−4.6 ± 0.4−17.3 ± 0.65.4 ± 0.36 ± 1.7α1A + α2 − δ + β3 + NCS-1 (n = 5)2.9 ± 0.7−4.9 ± 0.2−17.6 ± 1.15.6 ± 0.36 ± 2.1Current-voltage and inactivation curve parameters calculated from oocytes expressing the indicated combination of subunits, in the presence of 10 mm external Ca2+. See “Experimental Procedures” for details.a Statistically different from control without NCS-1 expressed (0.05 level). Open table in a new tab Table IIEffects of NCS-1 on Cav2.1 Ba2+ currentsBa2+currentsV actK actVinK inR inmV%α1A + α2 − δ (n = 4)4.6 ± 1.1−5.8 ± 0.2−13.8 ± 1.45.9 ± 0.90 ± 2.7α1A + α2 − δ + NCS-1 (n = 7)6.1 ± 1.2−6.3 ± 0.2−14.2 ± 0.86.5 ± 0.40 ± 2.7α1A + α2 − δ + β1 (n = 12)−6.0 ± 0.7−4.9 ± 0.3−25.3 ± 0.85.5 ± 0.116 ± 2.1α1A + α2 − δ + β1 + NCS-1 (n = 7)−5.0 ± 0.6−5.0 ± 0.2−25.0 ± 0.95.7 ± 0.216 ± 1.9α1A + α2 − δ + β2 (n = 7)−10.7 ± 0.9−3.1 ± 0.2−10.2 ± 1.35.4 ± 0.667 ± 1.7α1A + α2 − δ + β2 + NCS-1 (n = 5)−6.9 ± 1.2aStatistically different from control without NCS-1 expressed (0.05 level).−4.1 ± 0.2aStatistically different from control without NCS-1 expressed (0.05 level).−5.7 ± 0.8aStatistically different from control without NCS-1 expressed (0.05 level).6.4 ± 0.562 ± 1.7aStatistically different from control without NCS-1 expressed (0.05 level).α1A + α2 − δ + β3 (n = 7)−3.8 ± 0.7−4.6 ± 0.3−27.4 ± 0.65.6 ± 0.27 ± 0.8α1A + α2 − δ + β3 + NCS-1 (n = 8)−5.2 ± 0.6−4.5 ± 0.3−30.8 ± 1.56.1 ± 0.26 ± 1.0Current-voltage and inactivation curve parameters calculated from oocytes expressing the indicated combination of subunits, in the presence of 10 mm external Ba2+. See “Experimental Procedures” for details.a Statistically different from control without NCS-1 expressed (0.05 level). Open table in a new tab Current-voltage and inactivation curve parameters calculated from oocytes expressing the indicated combination of subunits, in the presence of 10 mm external Ca2+. See “Experimental Procedures” for details. Current-voltage and inactivation curve parameters calculated from oocytes expressing the indicated combination of subunits, in the presence of 10 mm external Ba2+. See “Experimental Procedures” for details. We then analyzed the effects of NCS-1 on Ba2+ current kinetics. CaV2.1 current inactivation could be approximated by an exponential decaying phase, best described using a fast (τ1) and a slow (τ2) component. None of these components appeared to be significantly affected by expression of the Ca2+-binding protein NCS-1 (Fig.4). This lack of effect was found for channels co-expressed with the β1 or the β2subunit and in the presence of either extracellular Ba2+ or Ca2+. Neither the time constants (τ1 and τ2) nor their respective amplitude (%τ2) were changed at all voltages examined (see Fig. 4). Moreover, no effect on channel activation and reactivation were observed upon co-expression of NCS-1, whether the CaV2.1 subunit was expressed with the α2-δ alone or with any of the four β subunits (not shown). Therefore, although both channel expression and channel properties seemed to be regulated by NCS-1 in a β subunit-specific manner, they appear to require different subunit arrangements,i.e. expression was modified when β1, β2, or β4 subunits were expressed, whereas modifications in channel properties were only recorded in the presence of the β2 subunit. To get some insight into the possible molecular determinants involved in NCS-1 effects, the same experiments, with the β2asubunit, were conducted using two mutants of NCS-1. NCS-1E120Q, with its third EF-hand disrupted, showed impaired Ca2+-dependent conformational changes (19Weiss J.L. Archer D.A. Burgoyne R.D. J. Biol. Chem. 2000; 275: 40082-40087Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) but was still able to bind cellular proteins. NCS-1G2A, a myristoylation-deficient mutant of NCS-1, relocalized NCS-1 from the perinuclear region to the cytosol (29O'Callaghan D.W. Ivings L. Weiss J.L. Ashby M.C. Tepikin A.V. Burgoyne R.D. J. Biol. Chem. 2002; 277: 14227-14237Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Co-expression of either NCS-1E120Q or NCS-1G2Ahad the same effect as wild-type NCS-1 on the current-voltage curve of the CaV2.1+α2-δ+β2Ca2+ channel (i.e. a small but significant positive shift of ∼5 mV; see Fig. 5B and Table III). They differed, however, in their effects on the inactivation curve. Although the NCS-1E120Q completely suppressed the effect of NCS-1 on the residual current (R in; see Fig. 5 and TableIII), co-expression of NCS-1G2A left this parameter unchanged but suppressed the shift in V ininduced by wild-type NCS-1. These two mutants also had different actions on current amplitudes. Indeed, whereas NCS-1E120Qreduced the Ba2+ current amplitude when compared with control currents, recorde" @default.
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• W1971742114 title "Down-regulation of Voltage-gated Ca2+ Channels by Neuronal Calcium Sensor-1 Is β Subunit-specific" @default.
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