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Neuronal Ca2+ Sensor-1/Frequenin Functions in an Autocrine Pathway Regulating Ca2+ Channels in Bovine Adrenal Chromaffin Cells

2000; Elsevier BV; Volume: 275; Issue: 51 Linguagem: Inglês

10.1074/jbc.m008603200

ISSN

1083-351X

Autores

Jamie L. Weiss, Deborah A. Archer, Robert D. Burgoyne,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

NCS-1/frequenin belongs to a family of EF-hand-containing Ca2+ sensors expressed mainly in neurons. Overexpression of NCS-1/frequenin has been shown to stimulate neurotransmitter release but little else is known of its cellular roles. We have constructed an EF-hand mutant, NCS-1(E120Q), as a likely dominant inhibitor of cellular NCS-1 function. Recombinant NCS-1(E120Q) showed an impaired Ca2+-dependent conformational change but could still bind to cellular proteins. Transient expression of this mutant, but not NCS-1, in bovine adrenal chromaffin cells increased non-L-type Ca2+ channel currents. Cells expressing NCS-1(E120Q) no longer responded effectively to the removal of autocrine purinergic/opioid inhibition of Ca2+ currents but still showed voltage-dependent facilitation. These data are consistent with the existence of both voltage-dependent and voltage-independent pathways for Ca2+ channel inhibition in chromaffin cells. Our results suggest a novel function for NCS-1 specific for the voltage-independent autocrine pathway that negatively regulates non-L-type Ca2+ channels in chromaffin cells. NCS-1/frequenin belongs to a family of EF-hand-containing Ca2+ sensors expressed mainly in neurons. Overexpression of NCS-1/frequenin has been shown to stimulate neurotransmitter release but little else is known of its cellular roles. We have constructed an EF-hand mutant, NCS-1(E120Q), as a likely dominant inhibitor of cellular NCS-1 function. Recombinant NCS-1(E120Q) showed an impaired Ca2+-dependent conformational change but could still bind to cellular proteins. Transient expression of this mutant, but not NCS-1, in bovine adrenal chromaffin cells increased non-L-type Ca2+ channel currents. Cells expressing NCS-1(E120Q) no longer responded effectively to the removal of autocrine purinergic/opioid inhibition of Ca2+ currents but still showed voltage-dependent facilitation. These data are consistent with the existence of both voltage-dependent and voltage-independent pathways for Ca2+ channel inhibition in chromaffin cells. Our results suggest a novel function for NCS-1 specific for the voltage-independent autocrine pathway that negatively regulates non-L-type Ca2+ channels in chromaffin cells. neuronal Ca2+ sensor phosphate-buffered saline containing 0.1% Triton X-100 and 0.3% BSA 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid green fluorescent protein Calmodulin has been well characterized as a ubiquitously expressed Ca2+ sensor involved in the regulation of many cellular processes. Other EF-hand-containing proteins have been identified that are, in contrast, highly expressed predominantly in neurons (1Braunewell K.-H. Gundelfinger E.D. Cell Tissue Res. 1999; 295: 1-12Crossref PubMed Scopus (239) Google Scholar). Members of this neuronal Ca2+ sensor (NCS)1 family include proteins expressed only in the retina (e.g. recoverin (2Ray S. Zozulya S. Niemi G.A. Flaherty K.M. Brolley D. Dizhoor A.M. Mckay D.B. Hurley J.B. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5705-5709Crossref PubMed Scopus (97) Google Scholar)) and others expressed in neuronal and neuroendocrine cells such as the neurocalcins (3Okazaki K. Watanabe M. Ando Y. Hagiwara M. Terasawa M. Hidaka H. Biochem. Biophys. Res. Commun. 1992; 185: 147-153Crossref PubMed Scopus (81) Google Scholar, 4Ladant D. J. Biol. Chem. 1995; 270: 3179-3185Abstract Full Text Full Text PDF PubMed Google Scholar, 5Faurobert E. Chen C.K. Hurley J.B. Teng D.H. J. Biol. Chem. 1996; 271: 10256-10262Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and NCS-1 (6Nef S. Fiumelli H. de Castro E. Raes M.-B. Nef P. J. Receptor Signal Trans. 1995; 15: 365-378Crossref PubMed Scopus (60) Google Scholar) (otherwise known as frequenin (7Pongs O. Lindemeier J. Zhu X.R. Theil T. Endelkamp D. Krah-Jentgens I. Lambrecht H.-G. Koch K.W. Schwemer J. Rivosecchi R. Mallart A. Galceran J. Canal I. Barbas J.A. Ferrus A. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (283) Google Scholar, 8Olafsson P. Soares H.D. Herzog K.-H. Wang T. Morgan J.I. Lu B. Mol. Brain Res. 1997; 44: 73-82Crossref PubMed Scopus (50) Google Scholar, 9Olafsson P. Wang T. Lu B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8001-8005Crossref PubMed Scopus (87) Google Scholar)). The neuronal Ca2+-sensor proteins are 22 kDa, high-affinity Ca2+-binding proteins with more than 50% sequence identity (1Braunewell K.-H. Gundelfinger E.D. Cell Tissue Res. 1999; 295: 1-12Crossref PubMed Scopus (239) Google Scholar). The functional roles of the neuronally expressed members of the family are generally unknown but there are some clues as to the function of NCS-1. Overexpression of NCS-1 results in enhancement of evoked exocytosis in neurons (7Pongs O. Lindemeier J. Zhu X.R. Theil T. Endelkamp D. Krah-Jentgens I. Lambrecht H.-G. Koch K.W. Schwemer J. Rivosecchi R. Mallart A. Galceran J. Canal I. Barbas J.A. Ferrus A. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (283) Google Scholar, 9Olafsson P. Wang T. Lu B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8001-8005Crossref PubMed Scopus (87) Google Scholar, 10Rivosecchi R. Pongs O. Theil T. Mallart A. J. Physiol. 1994; 474: 223-232Crossref PubMed Scopus (78) Google Scholar) and neuroendocrine cells (11McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The basis of its effect on exocytosis is not known nor is it known whether it has other physiological functions. It is known, however, that multiple binding targets for NCS-1 can be detected in biochemical assays (12Schaad N.C. De Castro E. Nef S. Hegi S. Hinrichsen R. Martone M.E. Ellisman M.H. Sikkink R. Sygush J. Nef P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9253-9258Crossref PubMed Scopus (95) Google Scholar, 13McFerran B.W. Weiss J. Burgoyne R.D. J. Biol. Chem. 1999; 274: 30258-30265Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) suggesting diverse regulatory roles for NCS-1/frequenin. Surprisingly, a yeast frequenin exists which is essential for viability due to its requirement as a stimulator of PIK1, a yeast phosphatidylinositol-4-OH kinase (14Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nature Cell Biology. 1999; 1: 234-241Crossref PubMed Scopus (221) Google Scholar). The targets and functions of other neuronal members of this family are less well known. The enhancement of exocytosis due to overexpression of NCS-1 in PC12 cells was observed in intact cells following stimulation with the purinergic agonist ATP but not in digitonin-permeabilized cells when exocytosis was directly stimulated by Ca2+ (11McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13McFerran B.W. Weiss J. Burgoyne R.D. J. Biol. Chem. 1999; 274: 30258-30265Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These data suggest an indirect effect of NCS-1 on the regulation of exocytosis perhaps due to changes in receptors, channel function, or Ca2+ signaling. The first observed phenotype due to overexpression of frequenin inDrosophila was a neurotransmission abnormality (15Tanouye M.A. Ferrus A. Fujita S.C. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6548-6552Crossref PubMed Scopus (129) Google Scholar) reminiscent of the consequences of K+ channel dysfunction in shaker mutants due to mutations in K+ channel subunits. Recent work has demonstrated that calmodulin is a constitutive subunit and Ca2+-dependent regulator of both voltage-gated Ca2+ channels (16Zuhlke R.D. Pitt G.S. Deisseroth K. Tsien R.Y. Reuter H. Nature. 1999; 399: 159-162Crossref PubMed Scopus (756) Google Scholar, 17Lee A. Wong S.T. Gallagher D. Li B. Storm D.R. Scheuer T. Catterall W.A. Nature. 1999; 399: 155-159Crossref PubMed Scopus (1009) Google Scholar, 18Qin N. Olcese R. Bransby M. Lin T. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2435-2438Crossref PubMed Scopus (253) Google Scholar, 19Peterson B.Z. DeMaria C.D. Yue D.T. Neuron. 1999; 22: 549-558Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar) and Ca2+-activated K+ channels (20Fanger C.M. Ghanshani S. Logsdon N.J. Rauer H. Kalman K. Zhou J. Beckingham K. Chandy K.G. Cahalan M.D. Aiyar J. J. Biol. Chem. 1999; 274: 5746-5754Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). It is not known whether NCS-1 or related family members regulate channel function but the more distant KChIP Ca2+-binding proteins have recently been found to be required for normal activity of A-type K+ channels (21An W.F. Bowlby M.R. Bett M. Cao J. Ling H.P. Mendoza G. Hinson J.W. Mattsson K.I. Strassle B.W. Trimmer J.S. Rhodes K.J. Nature. 2000; 403: 553-556Crossref PubMed Scopus (845) Google Scholar). The aims of this project were 2-fold. First, to develop a novel EF-hand mutant of NCS-1 as a dominant inhibitor of NCS-1 function. Second, to further resolve the cellular functions of NCS-1. We have investigated its role in the regulation of Ca2+ currents in adrenal chromaffin cells and from the use of an EF-hand mutant we establish that NCS-1 acts in a physiological pathway for negative regulation of voltage-activated Ca2+channels. Plasmids encoding wild-type NCS-1 for protein expression (pET-5a/NCS-1) or in pcDNA3.1 for transfection (pNCS-1) were described previously (11McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13McFerran B.W. Weiss J. Burgoyne R.D. J. Biol. Chem. 1999; 274: 30258-30265Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Plasmids encoding NCS-1(E120Q) were prepared by site-directed mutagenesis using a QuikchangeTMsite-directed mutagenesis kit (Stratagene Europe, Amsterdam, The Netherlands). The sense and antisense primers used for mutagenesis (with changes underlined) and with a unique AccI restriction site were: 5′-CACCAGAAACCAGATGCTGGACATAGTCGACGCCATTTACC-3′ and 5′-GGTAAATGGCGTCGACTATGTCCAGCATCTGGTTTCTGGTG-3′, respectively. Plasmid sequences were confirmed by automated DNA sequencing. BL21(DE3) cells (Promega, Southampton, United Kingdom) were transformed with pBB131 (25Graham M.E. Burgoyne R.D. J. Neurosci. 2000; 20: 1281-1289Crossref PubMed Google Scholar), a plasmid encoding N-myristoyltransferase (NMT:myristoyltransferase-CoA protein N-myristoyl transferase (EC 2.3.1.97), and subsequently with pET-5a/NCS-1 or pET-5a/NCS-1(E120Q) constructs. Expression of both recombinant NCS-1 and NMT was induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside at 37 °C for 4 h in the presence of 5 μg/ml myristate. Cells were harvested, lysed, and recombinant NCS-1 or NCS-1(E120Q) purified as described previously (13McFerran B.W. Weiss J. Burgoyne R.D. J. Biol. Chem. 1999; 274: 30258-30265Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and stored at −80 °C. For mass spectroscopy analysis, protein was concentrated using Nanosep 10K centrifugal concentrators (Pall Gelman Laboratory, Portsmouth, UK) and dialyzed against distilled H2O with Slide-A-LyzerTM 10K dialysis cassettes (Pierce and Warriner, Chester, UK). NCS-1 and NCS-1(E120Q) were biotinylated and used to probe immobilized adrenal membrane and cytosol fractions on nitrocellulose blots in the presence of 10 μm free Ca2+ as described previously (13McFerran B.W. Weiss J. Burgoyne R.D. J. Biol. Chem. 1999; 274: 30258-30265Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Samples for SDS-polyacrylamide gel electrophoresis were dissolved in sample buffer (125 mm HEPES (pH 6.8), 1.25% SDS (w/v), 2 mmEDTA, 10% sucrose (w/v), 1% β-mercaptoethanol, 10% glycerol (v/v), and 0.001% bromphenol blue), boiled, and separated on a 10 or 15% SDS-polyacrylamide gels. For experiments investigating changes in protein conformation, samples were prepared in non-denaturing sample buffer (50 mm Trizma base, 10% glycerol, 10% sucrose, 0.001% bromphenol blue, pH 7.5, with either 10 mm EGTA or 10 mm CaCl2), boiled, and separated on a 10% polyacrylamide gel in the absence of SDS. Freshly isolated bovine adrenal chromaffin cells from adult cows (22Burgoyne R.D. Boulton A. Baker G. Taylor C. Neuromethods: Intracellular Messengers. Humana Press Inc., Totowa, NJ1992: 433-470Google Scholar) were plated on non-tissue culture-treated 10-cm Petri dishes at a density of 1 × 106/ml. The following day non-attached cells were harvested by centrifugation, re-suspended in growth medium at a density of 1 × 107/ml and 20 μg of pEGFP (CLONTECH, Basingstoke, UK) in combination with 20 μg of pDNA3.1 (control cells), pNCS-1, or pNCS-1(E120Q) was added per ml of cells. Cells and plasmids were electroporated at 250 V and 975 microfarads for one pulse, using a Bio-Rad Gene Pulser II (Bio-Rad) and 4-mm cuvettes. Cells were diluted as rapidly as possible to 1 × 106/ml with fresh growth media and 1 × 106 cells grown on 35-mm Petri dishes in a final volume of 3 ml of growth medium for a further 3–5 days. Transfections were performed as above except that the cells were plated onto round glass coverslips (13 mm diameter). Afer washing twice with phosphate-buffered saline, cells were fixed in 3.7% formaldehyde in phosphate-buffered saline for 2 h at room temperature. The cells were then washed twice in phosphate-buffered saline, incubated for 30 min in PBT, and incubated for 2 h with rabbit anti-NCS-1 (11McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) at a 1/400 dilution in PBT. After washing three times in PBT, cells were incubated for 1 h in biotinylated anti-rabbit IgG (Amersham Pharmacia Biotech) at 1/100 in PBT, washed three times with PBT, and finally incubated in streptavidin/Texas Red (Amersham Pharmacia Biotech) at 1/50 dilution in PBT for 30 min. The cells were viewed with the appropriate filters for GPF fluorescence and immunofluorescence. Adrenal chromaffin cells grown on 35-mm plastic dishes were washed and maintained in external bath solution. For recording of Ca2+ channel currents (23Twitchell W.A. Rane S.G. Neuron. 1993; 10: 701-709Abstract Full Text PDF PubMed Scopus (51) Google Scholar), this contained 143 mm tetraethylammonium chloride, 10 mm BaCl2, 1 mm MgCl2, and 10 mm HEPES, pH 7.3. The intracellular pipette solution contained 150 mm CsCl, 2 mm Mg-ATP, 2 mm BAPTA, 0.5 mm GTP, and 10 mmHEPES, pH 7.3. The pipettes used had a resistance of 2.0–4.0 MΩ in the bath solution. Whole cell recording was carried out using an EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht, Germany). The cells were held at −60 mV after correction for a liquid junction potential of −8mV and depolarized under the control of PULSE software (HEKA). Capacitance and series resistance were compensated using the EPC-9 correction routines. Series resistance was on average less than 10 MΩ. Current output was filtered at 2.9 KHz with a 4-pole Bessel filter and data acquired and stored at 20-μs intervals. Linear leak and capacity currents were subtracted with a P/4 procedure using PULSE software and traces are the averages of three sweeps. All experiments were carried out at room temperature (22–24 °C) without superfusion during recording. All drug applications were by direct addition to the bath buffer. The recent establishment of calmodulin as a regulator of Ca2+ and K+ channel function has been based on co-expression of channel subunits and calmodulin constructs with inactivating mutations in one or more EF-hands (16Zuhlke R.D. Pitt G.S. Deisseroth K. Tsien R.Y. Reuter H. Nature. 1999; 399: 159-162Crossref PubMed Scopus (756) Google Scholar, 19Peterson B.Z. DeMaria C.D. Yue D.T. Neuron. 1999; 22: 549-558Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar, 20Fanger C.M. Ghanshani S. Logsdon N.J. Rauer H. Kalman K. Zhou J. Beckingham K. Chandy K.G. Cahalan M.D. Aiyar J. J. Biol. Chem. 1999; 274: 5746-5754Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). These mutants act in a dominant negative fashion to block the function of endogenous calmodulin. Such an approach could also work for NCS-1 as it binds to many of its target proteins in a Ca2+-independent manner (13McFerran B.W. Weiss J. Burgoyne R.D. J. Biol. Chem. 1999; 274: 30258-30265Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). NCS-1 possesses four EF-hands with the first N-terminal EF-hand being non-functional (24Cox J.A. Drussel I. Comte M. Nef S. Nef P. Lenz S.E. Gundelfinger E.D. J. Biol. Chem. 1994; 269: 32807-32814Abstract Full Text PDF PubMed Google Scholar). In order to develop a probe for NCS-1 function, we constructed an EF-hand mutant of NCS-1 with glutamate at position 120 mutated to glutamine (E120Q) in the third EF-hand (Fig. 1 a). This was based on a mutation previously characterized in the related protein neurocalcin δ and shown to impair Ca2+ binding (4Ladant D. J. Biol. Chem. 1995; 270: 3179-3185Abstract Full Text Full Text PDF PubMed Google Scholar). NCS-1(E120Q) was initially expressed as a recombinant protein and purified as described previously for wild-type NCS-1 (13McFerran B.W. Weiss J. Burgoyne R.D. J. Biol. Chem. 1999; 274: 30258-30265Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) (Fig. 1 b). Following co-expression in Escherichia coli withN-myristoyltransferase, both wild-type NCS-1 and NCS-1(E120Q) were N terminally myristoylated to around 20% based on mass spectroscopy. We examined whether NCS-1(E120Q) had an impairment of its Ca2+-dependent function. Ca2+ binding leads to a conformational change in the neuronal Ca2+ sensor proteins which can be assayed as a change in migration during native polyacrylamide gel electrophoresis in the presence of Ca2+ (5Faurobert E. Chen C.K. Hurley J.B. Teng D.H. J. Biol. Chem. 1996; 271: 10256-10262Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Wild-type NCS-1 showed a marked change in migration in native gel electrophoresis in the presence of Ca2+ compared with the presence of EGTA (Fig. 1 c). The change in migration of NCS-1(E120Q) due to Ca2+ was much less than for the wild-type protein. These data show that the E120Q mutation impairs the normal conformational change in NCS-1 occurring upon Ca2+ binding and suggests that this mutant protein is functionally impaired. If NCS-1(E120Q) is to act in a dominant negative manner it would have to be able to bind to target proteins. Using a biotinylated protein overlay technique, previously used to identify target proteins for NCS-1(13), we found that NCS-1(E120Q) binds the same pattern of polypeptides in adrenal membrane and cytosol fractions as seen for NCS-1 (Fig. 1 d). These results suggest that NCS-1(E120Q) expressed in cells could act in a dominant negative manner to interfere with the function of endogenous NCS-1.Figure 1Preparation and characterization of an EF-hand mutant of NCS-1. a, schematic diagram of the EF-hand domains in NCS-1 showing the introduced mutation at the nucleotide (G → C) and protein (E120Q) level in the third EF-hand.b, SDS-polyacrylamide gel electrophoresis of purified recombinant NCS-1 and NCS-1(E120Q). c, analysis of the migration of NCS-1 and NCS-1(E120Q) on native polyacrylamide gel electrophoresis. The arrows on the left andright show the shift in migration due to Ca2+ in NCS-1 and NCS-1(E120Q), respectively. d, identification of NCS-1- and NCS-1(E120Q)-binding proteins in chromaffin membrane (M) and cytosol (C) fractions from biotinylated protein overlays in the presence of 10 μm free Ca2+.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Bovine adrenal chromaffin cells were transiently transfected and analyzed by whole cell patch clamp recording as these cells express NCS-1 (11McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). To identify the small proportion of successfully transfected cells for recording, the cells were co-transfected with a plasmid encoding green fluorescent protein (GFP) along with a plasmid encoding either NCS-1 or NCS-1(E120Q). This approach has previously been shown to lead to high levels (>95%) of co-expression in transfected adrenal chromaffin cells (25Graham M.E. Burgoyne R.D. J. Neurosci. 2000; 20: 1281-1289Crossref PubMed Google Scholar, 26Graham M.E. Fisher R.J. Burgoyne R.D. Biochimie (Paris ). 2000; 82: 469-479Crossref PubMed Scopus (84) Google Scholar). Expression of wild-type or mutated NCS-1 following transfection was verified by immunofluorescent staining. Using a concentration of anti-NCS-1 antiserum (1:400) too low to stain endogenous NCS-1, it was observed that GFP-negative cells showed no more than background staining. In contrast, GFP-positive cells were brightly stained showing that they overexpressed wild-type NCS-1 or NCS-1(E120Q) following transfection (Fig. 2). Essentially all GFP-positive cells were stained above background by anti-NCS-1 after transfection with plasmids encoding NCS-1 or NCS-1(E120Q). Ca2+ channel currents were monitored in GFP-expressing cells by whole cell voltage-clamp recording using standard protocols (23Twitchell W.A. Rane S.G. Neuron. 1993; 10: 701-709Abstract Full Text PDF PubMed Scopus (51) Google Scholar) with Ba2+ as the charge carrier. Cells transfected with GFP and pcDNA3.1 vector were used as controls. These control cells showed no obvious differences from non-transfected cells in their electrophysiological characteristics and possessed maximal Ca2+ currents of 235.4 ± 60pA (n = 10) on depolarization from a holding potential of −60 to 0 mV. Ca2+-channel currents monitored in control (GFP-expressing) cells and cells expressing NCS-1 or NCS-1(E120Q) were averaged for all cells from each condition. In each case, the currents were non-inactivating as shown for a depolarization step to 0 mV (Fig. 3 a). The averaged currents from NCS-1-overexpressing cells were little different from control cells. In contrast, a marked increase in Ca2+-channel currents was observed in cells expressing NCS-1(E120Q). Despite the difference in current magnitude, little difference was seen between conditions in the initial kinetics of current activation when the currents were normalized (Fig. 3 b). For more detailed comparison of the magnitude of Ca2+-channel currents, the values were corrected for cell capacitance to eliminate differences due to cell size. It was clear from these data that overexpression of wild-type NCS-1 had no significant effect on the size of Ca2+ currents in transfected chromaffin cells or their current-voltage relationship (Fig. 3 c). In contrast, cells transfected with NCS-1(E120Q) showed a large increase in Ca2+ currents (significant from analysis of variance,p < 0.0001). No changes were observed in the current/voltage relationships of the Ca2+ currents measured following expression of NCS-1(E120Q) (Fig. 3 c). Most studies have found that bovine chromaffin cells possess predominantly non-L-type Ca2+channels (27Albillos A. Garcia A.G. Gandia L. FEBS Lett. 1993; 336: 259-262Crossref PubMed Scopus (74) Google Scholar, 28Currie K.P.M. Fox A.P. Neuron. 1996; 16: 1027-1036Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) but it has been controversially claimed that L-type Ca2+ channels can be recruited by a variety of manipulations (29Artalejo C.R. Ariano M.A. Perlman R.L. Fox A.P. Nature. 1990; 348: 239-242Crossref PubMed Scopus (193) Google Scholar, 30Artalejo C.R. Adams M.E. Fox A.P. Nature. 1994; 367: 72-76Crossref PubMed Scopus (242) Google Scholar). The effect of the L-type Ca2+channel blocker nifedipine on Ca2+ currents in NCS1-(E120Q)-expressing cells was examined. The Ca2+currents were found to be predominantly non-L-type as 3 μm nifedipine resulted in only between 0 and 18% inhibition of currents whereas these were abolished by addition of 250 μm cadmium (n = 4 cells). Under the conditions used here, non-L-type Ca2+ currents are under tonic autocrine inhibition (31Albillos A. Gandia L. Michelena P. Gilabert J.A. del Valle M. Carbone E. Garcia A.G. J. Physiol. 1996; 494: 687-695Crossref PubMed Scopus (67) Google Scholar). This is due to endogenous release of ATP and opioids from chromaffin cells in the dish. This inhibition can be removed by addition of P2y purinergic and opioid receptor antagonists (28Currie K.P.M. Fox A.P. Neuron. 1996; 16: 1027-1036Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 31Albillos A. Gandia L. Michelena P. Gilabert J.A. del Valle M. Carbone E. Garcia A.G. J. Physiol. 1996; 494: 687-695Crossref PubMed Scopus (67) Google Scholar, 32Carabelli V. Carra I. Carbone E. Neuron. 1998; 20: 1255-1268Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In order to examine the relationship between the additional Ca2+ current in NCS1-(E120Q)-expressing cells and the recruitment of this pool of quiet Ca2+ channels, we examined the effect of addition of a combination of 100 μm suramin and 10 μmnaloxone to block purinergic and opioid receptors, respectively. The antagonists (Fig. 4 a) resulted in a rapid increase in Ca2+ currents in all 7 control cells with an increase of >20% in 6 cells. Under our conditions, addition of naloxone/suramin did not modify the channel activation kinetics. In contrast, addition of the antagonists to cells expressing NCS-1(E120Q) resulted in a much reduced increase in Ca2+ currents (Fig. 4 b) with an increase of <10% in 6 of 8 cells. The difference between the cells is shown in a comparison of the mean increases in Ca2+ current observed (Fig. 4 c). The effect of NCS-1(E120Q) cannot be due to an inhibition of secretion of ATP and opioids in the cultures as this occurs from all of the non-voltage-clamped cells in the dish of which we estimated that only 2% were transfected. The autocrine inhibition of Ca2+ channels in chromaffin cells can be overcome, but only partially, by prior strong depolarization (facilitation) suggesting the presence of both voltage-dependent and -independent mechanisms for channel inhibition (27Albillos A. Garcia A.G. Gandia L. FEBS Lett. 1993; 336: 259-262Crossref PubMed Scopus (74) Google Scholar, 28Currie K.P.M. Fox A.P. Neuron. 1996; 16: 1027-1036Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Two distinct receptor-dependent pathways for channel inhibition that differ in their reversibility by prior depolarization have been described for various neuronal cell types (33Diverse-Pierluissi M. Goldsmith P.K. Dunlap K. Neuron. 1995; 14: 191-200Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 34Formenti A. Martina M. Plebani A. Mancia M. J. Physiol. 2000; 509: 395-409Crossref Scopus (38) Google Scholar, 35Dunlap K. Ikeda S.R. Semin. Neurosci. 1998; 9: 198-208Crossref Scopus (15) Google Scholar, 36Arnot M.I. Stotz S.C. Jarvis S.E. Zamponi G.W. J. Physiol. 2000; 527: 203-212Crossref PubMed Scopus (59) Google Scholar, 37Delmas P. Abogadie F.C. Dayrell M. Haley J.E. Milligan G. Caulfield M.P. Brown D.A. Buckley N.J. Eur. J. Neurosci. 1998; 10: 1654-1666Crossref PubMed Scopus (71) Google Scholar). To test which type of pathway was blocked by NCS-1(E120Q), Ca2+ currents were examined before and after a 50-ms prepulse to +90 mV. Facilitation was observed in control cells (Fig. 4 d) and a similar extent of facilitation was seen in NCS-1(E120Q)-expressing cells (Fig. 4, e and f). Voltage-dependent facilitation has been shown to increase the rate of channel activation as well as increasing steady-state current magnitude (33Diverse-Pierluissi M. Goldsmith P.K. Dunlap K. Neuron. 1995; 14: 191-200Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 34Formenti A. Martina M. Plebani A. Mancia M. J. Physiol. 2000; 509: 395-409Crossref Scopus (38) Google Scholar, 35Dunlap K. Ikeda S.R. Semin. Neurosci. 1998; 9: 198-208Crossref Scopus (15) Google Scholar, 36Arnot M.I. Stotz S.C. Jarvis S.E. Zamponi G.W. J. Physiol. 2000; 527: 203-212Crossref PubMed Scopus (59) Google Scholar, 37Delmas P. Abogadie F.C. Dayrell M. Haley J.E. Milligan G. Caulfield M.P. Brown D.A. Buckley N.J. Eur. J. Neurosci. 1998; 10: 1654-1666Crossref PubMed Scopus (71) Google Scholar). From examination of normalized traces it was clear that neither expression of NCS-1(E120Q) (Fig. 3 b) nor addition of naloxone and suramin (Fig. 5 a) modified the activation kinetics of the Ca2+ channel currents. In contrast, the prepulse protocol in either control or NCS-1(E120Q)-expressing cells produced a small but consistent increase in the rate of Ca2+ channel activation (Fig. 5, b andc). We also examined whether prepulse facilitation could still be elicited following addition of naloxone and suramin in control cells. In 4 cells tested an increase in current was seen following naloxone and suramin addition and in all 4 cells prepulse facilitation occurred to essentially the same extent as before antagonist addition (Fig. 6). These data are consistent with the existence of two distinct pathways for Ca2+ channel inhibition in chromaffin cells and with the NCS-1 mutant acting via the voltage-independent pathway. The results presented here suggest that NCS-1/frequenin may be a physiological regulator of Ca2+ channels in adrenal chromaffin cells based on the use of an EF-hand mutant, NCS-1(E120Q), that we suggest acts in a dominant negative manner to disrupt the function of endogenous NCS-1. Overexpression of wild-type NCS-1 had no effect on Ca2+ channel currents in chromaffin cells, while expression of NCS-1(E120Q) resulted in an increase in non-L-type Ca2+ channel currents. The differences in the effect of these two proteins demonstrated the specificity of these changes and that the NCS-1(E120Q) effect was not just the consequence of protein overexpression. It was also not likely to be due to any deleterious effects of NCS-1(E120Q) expression on the cells. This would instead, be more likely to reduce Ca2+ channel currents as their maintenance is well known to be dependent on high intracellular ATP levels. We would argue that the effect of NCS-1(E120Q) on Ca2+ channel currents is a consequence of a dominant negative effect of this EF-hand mutant as seen for analogous calmodulin mutants (16Zuhlke R.D. Pitt G.S. Deisseroth K. Tsien R.Y. Reuter H. Nature. 1999; 399: 159-162Crossref PubMed Scopus (756) Google Scholar, 19Peterson B.Z. DeMaria C.D. Yue D.T. Neuron. 1999; 22: 549-558Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar, 20Fanger C.M. Ghanshani S. Logsdon N.J. Rauer H. Kalman K. Zhou J. Beckingham K. Chandy K.G. Cahalan M.D. Aiyar J. J. Biol. Chem. 1999; 274: 5746-5754Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Overexpression of wild-type calmodulin also has no effects on the activity of calmodulin-regulated channels. While we saw no effect of overexpression of NCS-1 in chromaffin cells our interpretation is supported by a description of an inhibition of high voltage-activated currents by overexpression of NCS-1/frequenin in neuroblastoma NG108–15 cells (38Burley J.R. Jeromin A. Robertson B. Roder J.C. Sihra T.S. Biophys. J. 2000; 78: 611Google Scholar). Together these observations suggest that NCS-1 normally functions in a pathway that inhibits high voltage-activated Ca2+ channels. The major effect that we observed in this study was an increase in Ca2+ channel current magnitude in cells expressing NCS-1(E120Q) with no effect on the kinetics of activation, inactivation or current voltage relationship of the currents consistent with a recruitment of additional functional channels without modification of channel properties. The role of NCS-1 in inhibiting Ca2+channel function is opposite to that which would explain the stimulatory effects of NCS-1/frequenin overexpression on neurotransmitter release (7Pongs O. Lindemeier J. Zhu X.R. Theil T. Endelkamp D. Krah-Jentgens I. Lambrecht H.-G. Koch K.W. Schwemer J. Rivosecchi R. Mallart A. Galceran J. Canal I. Barbas J.A. Ferrus A. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (283) Google Scholar, 9Olafsson P. Wang T. Lu B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8001-8005Crossref PubMed Scopus (87) Google Scholar, 11McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). We have identified, therefore, a novel, alternative cellular function for this protein that appears to be distinct from its effect on neurotransmitter release. It is likely that further use of the NCS-1(E120Q) mutant and similar constructs for other members of this Ca2+-binding protein family will allow discovery of other novel physiological roles of these proteins. Bovine adrenal chromaffin cells possess mainly non-L-type Ca2+ channels (27Albillos A. Garcia A.G. Gandia L. FEBS Lett. 1993; 336: 259-262Crossref PubMed Scopus (74) Google Scholar) and it has been well established (31Albillos A. Gandia L. Michelena P. Gilabert J.A. del Valle M. Carbone E. Garcia A.G. J. Physiol. 1996; 494: 687-695Crossref PubMed Scopus (67) Google Scholar) that a substantial pool of these Ca2+ channels can be inhibited, in an autocrine manner, by endogenously released ATP and opioids (23Twitchell W.A. Rane S.G. Neuron. 1993; 10: 701-709Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 28Currie K.P.M. Fox A.P. Neuron. 1996; 16: 1027-1036Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 32Carabelli V. Carra I. Carbone E. Neuron. 1998; 20: 1255-1268Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 39Diverse-Pierluissi M. Dunlap K. Westhead E.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1261-1265Crossref PubMed Scopus (66) Google Scholar, 40Powell A.D. Teschemacher A.G. Seward E.P. J. Neurosci. 2000; 20: 606-616Crossref PubMed Google Scholar). They can, however, be recruited into a functional pool under various conditions (28Currie K.P.M. Fox A.P. Neuron. 1996; 16: 1027-1036Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 31Albillos A. Gandia L. Michelena P. Gilabert J.A. del Valle M. Carbone E. Garcia A.G. J. Physiol. 1996; 494: 687-695Crossref PubMed Scopus (67) Google Scholar, 32Carabelli V. Carra I. Carbone E. Neuron. 1998; 20: 1255-1268Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 41Doupnik C.A. Pun R.Y.K. Pflugers Arch. 1992; 420: 61-71Crossref PubMed Scopus (30) Google Scholar, 42Cena V. Stutzin A. Rojas E. J. Memb. Biol. 1989; 112: 255-265Crossref PubMed Scopus (41) Google Scholar). The inhibited pool consists of a complex mixture of all non-L-type channels in chromaffin cells including P/Q- and N-type. This regulatory pathway operates by pertussis toxin-sensitive, G-protein-dependent mechanisms (31Albillos A. Gandia L. Michelena P. Gilabert J.A. del Valle M. Carbone E. Garcia A.G. J. Physiol. 1996; 494: 687-695Crossref PubMed Scopus (67) Google Scholar, 41Doupnik C.A. Pun R.Y.K. Pflugers Arch. 1992; 420: 61-71Crossref PubMed Scopus (30) Google Scholar, 42Cena V. Stutzin A. Rojas E. J. Memb. Biol. 1989; 112: 255-265Crossref PubMed Scopus (41) Google Scholar) likely to involve, at least in part, direct interaction of G-protein βγ-subunits with Ca2+ channels (43Dolphin A.C. J. Physiol. 1998; 506: 3-11Crossref PubMed Scopus (236) Google Scholar, 44Herlitze S. Garcia D.E. Mackie K. Hille B. Scheuer T. Catterall W.A. Nature. 1996; 380: 258-262Crossref PubMed Scopus (714) Google Scholar). We revealed this inhibited current by addition of the purinergic and opioid antagonists suramin and naloxone as described previously (28Currie K.P.M. Fox A.P. Neuron. 1996; 16: 1027-1036Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 31Albillos A. Gandia L. Michelena P. Gilabert J.A. del Valle M. Carbone E. Garcia A.G. J. Physiol. 1996; 494: 687-695Crossref PubMed Scopus (67) Google Scholar, 32Carabelli V. Carra I. Carbone E. Neuron. 1998; 20: 1255-1268Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Following the increase in Ca2+current due to NCS-1(E120Q) expression, addition of the purinergic and opioid antagonists produced a substantially reduced increase in Ca2+ currents compared with control cells consistent with NCS-1(E120Q) and the antagonists acting on the same pool of Ca2+ channels. This suggests that the mechanism of the NCS-1(E120Q) effect was to remove the autocrine inhibition of Ca2+ channels in the cells. This implies that endogenous NCS-1 functions in this autocrine inhibitory pathway. In contrast to the results with the antagonists, the mutant did not inhibit facilitation due to a prepulse protocol. These findings are, however, consistent with previous work on chromaffin cells and on neuronal preparations. The autocrine inhibition of Ca2+ channel currents in chromaffin cells due to released secretory products is only reversed by up to 50% by pre-pulse depolarization suggesting the presence of two pathways, one voltage-dependent and one voltage-independent (28Currie K.P.M. Fox A.P. Neuron. 1996; 16: 1027-1036Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 31Albillos A. Gandia L. Michelena P. Gilabert J.A. del Valle M. Carbone E. Garcia A.G. J. Physiol. 1996; 494: 687-695Crossref PubMed Scopus (67) Google Scholar). In some neuronal cell types a single pathway, fully reversible by prior depolarization exists (43Dolphin A.C. J. Physiol. 1998; 506: 3-11Crossref PubMed Scopus (236) Google Scholar). In contrast, other neurons also show evidence for two distinct pathways (33Diverse-Pierluissi M. Goldsmith P.K. Dunlap K. Neuron. 1995; 14: 191-200Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 34Formenti A. Martina M. Plebani A. Mancia M. J. Physiol. 2000; 509: 395-409Crossref Scopus (38) Google Scholar, 35Dunlap K. Ikeda S.R. Semin. Neurosci. 1998; 9: 198-208Crossref Scopus (15) Google Scholar, 36Arnot M.I. Stotz S.C. Jarvis S.E. Zamponi G.W. J. Physiol. 2000; 527: 203-212Crossref PubMed Scopus (59) Google Scholar, 37Delmas P. Abogadie F.C. Dayrell M. Haley J.E. Milligan G. Caulfield M.P. Brown D.A. Buckley N.J. Eur. J. Neurosci. 1998; 10: 1654-1666Crossref PubMed Scopus (71) Google Scholar). These have been characterized in detail in dorsal root ganglion (33Diverse-Pierluissi M. Goldsmith P.K. Dunlap K. Neuron. 1995; 14: 191-200Abstract Full Text PDF PubMed Scopus (119) Google Scholar), rat sympathetic (37Delmas P. Abogadie F.C. Dayrell M. Haley J.E. Milligan G. Caulfield M.P. Brown D.A. Buckley N.J. Eur. J. Neurosci. 1998; 10: 1654-1666Crossref PubMed Scopus (71) Google Scholar), and rat sensory neurons (34Formenti A. Martina M. Plebani A. Mancia M. J. Physiol. 2000; 509: 395-409Crossref Scopus (38) Google Scholar), for example. One pathway leads to inhibition of Ca2+ channel current has no effect on activation kinetics is not reversed by prior depolarization (33Diverse-Pierluissi M. Goldsmith P.K. Dunlap K. Neuron. 1995; 14: 191-200Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 34Formenti A. Martina M. Plebani A. Mancia M. J. Physiol. 2000; 509: 395-409Crossref Scopus (38) Google Scholar, 35Dunlap K. Ikeda S.R. Semin. Neurosci. 1998; 9: 198-208Crossref Scopus (15) Google Scholar, 36Arnot M.I. Stotz S.C. Jarvis S.E. Zamponi G.W. J. Physiol. 2000; 527: 203-212Crossref PubMed Scopus (59) Google Scholar, 37Delmas P. Abogadie F.C. Dayrell M. Haley J.E. Milligan G. Caulfield M.P. Brown D.A. Buckley N.J. Eur. J. Neurosci. 1998; 10: 1654-1666Crossref PubMed Scopus (71) Google Scholar). The second pathway also results in a reduction in magnitude and also in slowing of activation kinetics, and is fully reversed by prepulse depolarization (i.e. is voltage-dependent). The two pathways involve different neurotransmitter receptors and G-proteins (33Diverse-Pierluissi M. Goldsmith P.K. Dunlap K. Neuron. 1995; 14: 191-200Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 35Dunlap K. Ikeda S.R. Semin. Neurosci. 1998; 9: 198-208Crossref Scopus (15) Google Scholar, 36Arnot M.I. Stotz S.C. Jarvis S.E. Zamponi G.W. J. Physiol. 2000; 527: 203-212Crossref PubMed Scopus (59) Google Scholar, 37Delmas P. Abogadie F.C. Dayrell M. Haley J.E. Milligan G. Caulfield M.P. Brown D.A. Buckley N.J. Eur. J. Neurosci. 1998; 10: 1654-1666Crossref PubMed Scopus (71) Google Scholar). NCS-1(E120Q) expression did not affect activation kinetics (Fig. 3 b) nor did it inhibit the facilitation due to prepulse depolarization. These results suggest, therefore, that chromaffin cells also possess two inhibitory pathways and are entirely consistent with a role of NCS-1 in only the voltage-independent pathway. In addition, the effect of prepulse depolarization must be to reverse inhibition due, not to ATP or opioids, but to some other component released from the secretory granules. Previous work has suggested the presence of several inhibitory components present is chromaffin cell secretory granules (31Albillos A. Gandia L. Michelena P. Gilabert J.A. del Valle M. Carbone E. Garcia A.G. J. Physiol. 1996; 494: 687-695Crossref PubMed Scopus (67) Google Scholar). In conclusion, the results presented here provide insight into a novel function for NCS-1 in which it acts on a receptor-mediated feedback pathway controlling non-L-type Ca2+ channel function in chromaffin cells. Our data suggest the existence of both voltage-dependent and voltage-independent pathways for the regulation of Ca2+ channels in chromaffin cells and that NCS-1 specifically acts in the voltage-independent pathway. It is possible that NCS-1 may play a similar role in neurons where it could act on related presynaptic autoreceptor pathways to modulate Ca2+ channel function and thereby neurotransmitter release.

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