Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9253-9258, August 1996 Neurobiology

Direct modulation of targets by the neuronal sensor NCS-1 (calmodulin-binding //neuronal calcium-binding / synthase/) NICOLAS C. SCHAAD*, EDOUARD DE CASTROt, SERGE NEFt, SARAH HEGIt, ROBERT HINRICHSENt, MARYANN E. MARTONE§, MARK H. ELLISMAN§, ROBERT SIKKINK$, FRANK RUSNAK$, JURGEN SYGUSHII, AND PATRICK NEFt** tDepartment of Biochemistry, Science II, University of Geneva, CH-1211 Geneva, Switzerland; *Division of Clinical Psychopharmacology, Institutions Universitaires de Psychiatrie, CH-1225 Geneva, Switzerland; I1Department of Biochemistry, University of Montreal, CP 6128, Station Centreville, Montreal, PQ, Canada H3C 3J7; tDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104-2092; §Microscopy and Imaging Resource, University of California at San Diego, La Jolla, CA 92093-0608; and ISection of Hematology Research and Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905 Communicated by Stephen R. Heinemann, The Salk Institute for Biological Studies, La Jolla, CA, May 14, 1996 (received for review January 15, 1996)

ABSTRACT Ca2+ and its ubiquitous intracellular recep- calcium sensor (9). NCS-1 inhibits phos- tor calmodulin (CaM) are required in the nervous system, phorylation in a calcium-dependent manner (8). In this assay, among a host of cellular responses, for the modulation of CaM is ineffective. Our previous work (10) indicated that several important and ion channels involved in NCS-1 binds only two Ca2+ ions with a high and synaptic efficacy and neuronal plasticity. Here, we report that with a higher affinity (-3 x 10-7 M) than CaM (-4-20 x 10-6 CaM can be replaced by the NCS-1 M), which binds four Ca2+ ions. We have also shown that both in vitro and in vivo. NCS-1 is a calcium binding protein NCS-1 changes its conformation, and most likely its activity, in with two Ca2+-binding domains that shares only 21% of a calcium- and magnesium-dependent manner (10). We there- homology with CaM. We observe that NCS-1 directly activates fore concluded that NCS-1 is not a Ca2+-buffering protein like two Ca2+/CaM-dependent enzymes (3':5'-cyclic nucleotide and , since it exhibits all the properties phosphodiesterase and protein calcineurin). Co- of a Ca2+ sensor that responds, like CaM, to Ca2+ fluctuations activation of by NCS-1 and CaM results by exposing hydrophobic surfaces in order to interact with its in a higher activity than with CaM alone. Moreover, NCS-1 is target(s). Since NCS-1 is not only present in photoreceptors, coexpressed with calcineurin and nitric oxide synthase in but also in various neurons throughout the entire nervous several neuron populations. Finally, injections of NCS-1 into system, we wondered what function NCS-1 might fulfill in calmodulin-defective cam' Paramecium partially restore wild- nonretinal neurons. type behavioral responses. With this highly purified prepara- Here we report that a highly purified preparation of NCS-1 tion of NCS-1, we have obtained crystals suitable for crystal- can substitute or potentiate calmodulin effects both in vitro and lographic structure studies. NCS-1, despite its very different in vivo. NCS-1 can directly activate CaM-dependent enzymes structure, distribution, and Ca2+-binding affinity as com- such as the PDE or the calcineurin, and pared with CaM, can substitute for or potentiate CaM func- is also capable of potentiating the activity of NOS. Coexpres- tions. Therefore, NCS-1 represents a novel protein capable of sion of NCS-1 with these enzymes is observed in several mediating multiple Ca2+-signaling pathways in the nervous neuron populations. In vivo, NCS-1 alone partially restores the system. wild-type behavior in calmodulin-defective cam' Paramecium mutants. In the nervous system, calmodulin (CaM) directly modulates cyclic nucleotide-gated channels in vision and olfaction (1-3), MATERIALS AND METHODS and activates the production of nitric oxide (NO) by NO synthases (NOS) (4, 5). Paradoxically, CaM also stimulates Purification and Enzymatic Assays. Recombinant chicken enzymes with antagonist activities-e.g., (CaMKI-II) NCS-1 (7), bovine Recoverin (11), and chicken Vilipl (12) versus (calcineurin), or cyclase (adenylate cy- were produced in Escherichia coli and affinity-purified on a clase) versus 3' :5'-cyclic nucleotide phosphodiesterase (PDE). phenyl-Sepharose CL-4B column to apparent homogeneity as These enzymes can act locally at the synapse or mediate more described (10) and used in the different enzymatic assays. general cellular responses involved in the regulation of tran- Crystals of NCS-1, shown in Fig. 3C, were grown using scription factors ( expression). CaM is a 148-aa protein conditions obtained from a sparse matrix crystallization screen highly conserved between species-e.g., 88% identity between (13), and were authenticated by SDS/PAGE. Silver staining of plant and human calmodulin proteins-that belongs to the the resulting electrophoretic migration pattern, shown in Fig. large superfamily of Ca2+-binding proteins (6). 3D, revealed a protein of 22 kDa (identical to the relative We have recently characterized a neuron-specific calcium molecular weight of NCS-1) present in crystals shown in Fig. sensor named NCS-1 (7). This 22-kDa protein has 46% identity 3C. The NCS-1 preparation is therefore highly pure since it with Recoverin and 21% with CaM. NCS-1 is also highly forms crystals. Bovine brain PDE was purchased from Sigma conserved in evolution since we characterized NCS-1 or- and was assayed in triplicate as described (14). Recombinant thologs in rat, chicken, mouse (100% identity), and in Caeno- calcineurin A (CNA) and calcineurin B (CNB) subunits were rhabditis elegans (75% identity) (8). One function of NCS-1, tested in vitro, appears to be similar to the retina-specific Abbreviations: CaM, calmodulin, NOS, nitric oxide synthase; PDE, phosphodiesterase; CNA, calcineurin A; CNB, calcineurin B. **To whom reprint requests should be addressed at: Department of The publication costs of this article were defrayed in part by page charge Biochemistry, Sciences II, University of Geneva, 30 quai Ernest payment. This article must therefore be hereby marked "advertisement" in Ansermet, CH-1211 Geneva 4, Switzerland. e-mail: Patrick.Nef@ accordance with 18 U.S.C. §1734 solely to indicate this fact. biochem.unige.ch. 9253 Downloaded by guest on September 30, 2021 9254 Neurobiology: Schaad et al. Proc. Natl. Acad. Sci. USA 93 (1996) purified and reconstituted as described (15). Bovine brain NCS-1 targets could overlap with CaM targets. We first tested NOS was affinity-purified as described (16) and assayed in for a possible effect of NCS-1 on purified PDE which is a triplicate by measuring the conversion of [3H] to well-characterized CaM target that regulates the intracellular [3H]citrulline as described (17) . second messenger levels of cyclic AMP and cyclic GMP (20, Western Blot Analysis. Chicken brain and eye were homog- 21). In this assay, recombinant NCS-1 was able to substitute for enized with a Teflon glass in 5 vol of buffer (20 mM Tris HCl, pH 7.4/0.1 mM phenylmethylsulfonyl fluoride/0.5 ,g of pep- CaM and to function as a direct PDE activator (Fig. 1A). statine per ml/1 mM EGTA/0.1 mM diisopropyl fluorophos- NCS-1 produced a large increase (3.8 ± 0.3 fold, n = 7) in PDE phate/2% SDS) and centrifuged at 30,000 x g for 30 min. The activity which was slightly smaller than with CaM alone (6- to eye supernatant was concentrated 10 times by precipitation 7-fold increase). The NCS-1-dependent activation of PDE was with 70% trichloroacetic acid. Samples were subjected to saturable and concentration-dependent (see below). NCS-1 SDS/PAGE in the presence of 1 mM EDTA, transferred onto itself had no endogenous PDE activity. nitrocellulose, blocked with 0.2% casein/0.033% polyvinylpyr- CaM-Dependent Phosphatase Calcineurin. We next as- rolidone, and incubated with an affinity-purified NCS-1 poly- sayed the phosphatase calcineurin for NCS-1 stimulation since clonal antibody (18) obtained from rabbit. Immunocomplexes its activity is essential for synaptic activity and is subject to dual were detected with anti-rabbit IgG- con- Ca2+ ion control mediated by both CaM and CNB (22, 23). A jugate, then visualized with the substrate system 5-bromo-4- functional calcineurin consists of two tightly associ- chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/ ated subunits (A and B), and is regulated in a reversible and NTB), or with the chemiluminescent substrate 3-(2'- spiroadamantane)-4-methoxy-4-(3"phosphoryloxy) phenyl- Ca2+-dependent fashion by CaM. CNA contains the 1,2-dioxetane (AMPPD). and belongs to the family of serine/threonine protein phos- Immunohistochemistry. Vibratome sections (50 ,tm) of phatases. The B subunit (CNB) is a Ca2+-binding protein with adult rat brain were labeled using the avidin/biotin/ four Ca2+-binding sites that possesses 35% identity with CaM peroxidase complex method using affinity-purified polyclonal and 30% with NCS-1. CNA contains distinct binding sites for antibodies against NCS-1 (18), a mouse monoclonal antibody CaM and CNB and neither can substitute for the other to against calcineurin (Transduction Laboratories, Lexington, potentiate CNA phosphatase activity (24). Using recombinant KY), and a rabbit polyclonal antibody against brain NOS forms of CNA and CNB (15), we observed that NCS-1 could (Transduction Laboratories). replace CaM but not CNB to stimulate the phosphatase Paramecium Injection. Using a micropipette, cells were activity (Fig. 1B). Thus, CNA alone had low phosphatase injected with "10 pl of a solution containing either NCS-1, activity that could be stimulated somewhat by either CNB or wild-type Paramecium calmodulin, or a control buffer as CaM The maximum occurred in the described (19). Cells were then placed in a Na+/tetraethyl- (Fig. 1B). activity presence ammonium chloride solution which causes backward swim- of both CNB and CaM. CNA in the presence of NCS-1 and ming (19). The effect on backward swimming was measured 4, CaM had about the same phosphatase activity as CNA in the 12, and 30 h after the injections. presence of CaM, indicating that NCS-1 could not replace CNB. However, NCS-1 and CNB potentiated the activity of CNA to within 70% of the maximum activity of CNA plus CNB RESULTS plus CaM. This activity was significantly higher that the activity CaM-Dependent PDE. Due to the rather limited number of of either CNA plus CNB or CNA plus CS-1 alone (Fig. 1B) and potential Ca2+-dependent pathways and enzymes in neurons, was saturable in NCS-1 (see below). A PDE B Calcineurin C NO Synthase 1oo

90 180-

80 160,

140 -F 70- X 120

t 50 't 10oo .2 40, b 80

30 60-

20 240 . 1

10 I. PDE PDE+CaM PDE*NCS-1 NCS.1 CNA CNA.CNS CNAICMA CNA4NCS1 CNAICt CNA4NcS-1 CNA4,N NOS NOS4CA NO4C.M NOS 4CWI +CdI 4NC.1 .NCS.1 4NCS.1

FIG. 1. Activation of CaM-dependent enzymes by NCS-1. (A) Activation of PDE activity by NCS-1. Affinity-purified bovine brain PDE (0.002 units) was assayed by using cAMP as the substrate. Mg2+ concentration was 1 mM, and free Ca2+ concentration was 100 xM. NCS-1 (1 ,uM) or CaM (0.1 ,uM; Sigma) was added where indicated for a 15-min period of incubation. Both proteins potentiated the activity of PDE (P < 0.0001). NCS-1 alone had no endogenous PDE activity. Data are the mean ± SEM of 7-10 determinations observed with NCS-1 or CaM batches. One hundred percent of PDE activity correspond to the hydrolysis of 0.01 ,umol of 3':5'-cAMP to 5' AMP per min per mg. (B) Activation of calcineurin phosphatase activity by NCS-1. CNA reconstituted in presence of 0.1 mM Ca2+ with CNB, CaM, and/or NCS-1 was assayed as described (15). CaM (0.1 j,M) or NCS-1 (1 jLM) was added where indicated. NCS-1 potentiated the basal activity of CNA plus CNB, and substituted for CaM in the activation of calcineurin. Data are the mean ± SEM of three determinations. One hundred percent of calcineurin activity correspond to 10.4 nmol of 32P released per min per mg. (C) Activation of NOS activity by NCS-1. NOS activity was determined in triplicate. Free Ca2+ concentration was 100 ,uM. NCS-1 (1 ,LM) or CaM (0.1 t,M) was added where indicated. NCS-1 could potentiate the stimulatory effect of CaM (P < 0.001). NCS-1 alone had no endogenous NOS activity. Data are the mean ± SEM of four determinations using NCS-1 or CaM samples. One hundred percent of NOS activity correspond to the conversion of 7.14 pmol of arginine to citrulline per hour per mg. Downloaded by guest on September 30, 2021 Neurobiology: Schaad et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9255 NOS. NO is synthesized by a family of NOSs (25) that play on PDE, since its enzymatic activity can be measured simul- various roles in the nervous system (26). The neuronal type I taneously at low Mg2+ and in the presence of various Ca2+ NOS is Ca2+/CaM dependent, and CaM binds reversibly to concentrations. Since the two binding sites of NCS-1 accom- NOS with a 1:1 stoichiometry (4). Since NCS-1 is present in modate either Ca2+ or Mg2+ [although the NCS-1 affinity for several neuron populations (7) we wondered whether NOS Mg2+ (_10-3 M) is rather low], we decreased the free Mg2+ activity was regulated by NCS-1. We therefore purified type I concentration to 3 x 10-7 M in the PDE assay. In the absence NOS from bovine and measured NOS activity in of Ca2+ the NCS-1-mediated enzymatic activity of PDE was the presence of CaM, NCS-1, or both. The data indicated that low. Addition of increasing concentrations of Ca2+ resulted in NCS-1 potentiates NOS activity but cannot totally replace an almost 3-fold stimulation (Fig. 2A Inset). The maximal CaM (Fig. 1C). Indeed, in the absence of CaM, NOS activity effect on NCS-1 was observed at -0.5-1 X 10-6 M free Ca2+, was not stimulated by NCS-1. However, in the presence of a whereas at higher Ca2+ concentrations, the NCS-1-mediated low CaM concentration (Fig. 1C), NCS-1 enhanced NOS Ca2+-dependent stimulation of PDE was slightly reduced. In activity at a much higher level than CaM alone (see below). In these low Mg2+ conditions, the basal level of NCS-1- this assay, ovalbumin had no effect on CaM-dependent NOS independent PDE was unaffected by Ca2+. activity. Potentiation of the NOS Activity by NCS-1. A low concen- NCS-1 Half-Maximal Effect. To further characterize the tration of CaM was necessary to observe stimulation of NOS stimulation of CaM-dependent enzymes by NCS-1, we mea- activity by NCS-1. Our results are in agreement with the sured the concentration of NCS-1 needed for half-maximal observations that CaM not only confers Ca2+ sensitivity on activation (EC50) of PDE (Fig. 24), calcineurin (Fig. 2B), and NOS but also serves as a critical for electron transfer NOS (Fig. 2C). All three enzyme activities were saturable with and catalysis (31) by promoting conformational changes within NCS-1, and the EC50 values were 210 ± 35 nM (mean ± SEM the flavoprotein and -binding domains of NOS. To offour experiments) with PDE, -350 nM with calcineurin, and investigate the dual role of CaM and NCS-1 on NOS stimu- '600 nM with NOS. For each enzyme, NCS-1 was less potent lation, we measured NOS activity at different CaM concen- than CaM since the EC50 values for CaM are 1-10 nM with trations in the presence or absence of NCS-1. In the absence PDE (28), 10 nM with calcineurin (29), and -3-5 nM with of CaM, NCS-1 does not affect NOS activity. Addition of CaM NOS (30). alone resulted in concentration-dependent basal NOS activa- The NCS-1 Effect Is Ca2+-dependent. To investigate the tion. Addition of NCS-1 (1 ,M) and varying concentrations of stimulation of NCS-1-dependent enzymes by Ca2 , we focused CaM resulted in a 2- to 4-fold potentiation of the basal NOS A PDE B Calcine)urin C NO Synthase

c 0 4. E 0 C) (U 2g. 3- 3.

1 10 0 1 2 3 [NCS-1] (pM) [NCS-1] (NM) [NCS-1] (pM)

FIG. 2. Activation of CaM-dependent targets as a function of NCS-1 concentration. (A) Activation of PDE activity as a function of NCS-1 concentration. The dose-response curve of NCS-1-dependent PDE activation was determined in triplicate as described in the legend of Fig. L4 using various NCS-1 concentrations. NCS-1 was able to saturate PDE activity (EC50 = 210 ± 35 nM, n = 4). one hundred percent of PDE activity (1-fold stimulation) corresponds to the hydrolysis of 0.01 ,umol of 3':5'-cAMP to 5' AMP per min per mg. (Inset) Activation of NCS-1-dependent PDE activity as a function of free Ca2+ concentration. PDE activity was measured in quintuplet using 0.3 JIM instead of 1 mM Mg2+ concentration in the reaction because NCS-1 can also be activated by high Mg2+ concentrations (Kng = 1 mM). Increase of free Ca2+ concentration markedly enhanced the potentiation effect of NCS-1 (upper trace) on PDE activity, whereas the NCS-1-independent basal activity of PDE (lower trace) was not affected by Ca2+. The free Ca2+ concentrations were calculated using the program COMICS (27). EGTA was 2 mM, and added Ca2+ was in the millimolar range to minimize the contribution of contaminating Ca2+. (B) Activation of calcineurin phosphatase activity as a function of NCS-1 concentration. The dose-response curve of NCS-1-dependent calcineurin phosphatase activation was determined at least in duplicate as described in the legend of Fig. 1B using various NCS-1 concentrations. NCS-1 was able to saturate calcineurin activity (ECso = 350 nM, n = 3). One hundred percent of calcineurin activity correspond to 8.09 nmol of 32P released per min per mg. (C) Activation of CaM-dependent NOS activity by NCS-1. The dose-response curve was determined as described in the legend of Fig. 1C using purified bovine brain NOS and various NCS-1 concentrations. NCS-1 was able to saturate NOS activity in a concentration-dependent manner (EC50 = 600 nM). CaM concentration was 0.1 ,uM, and free Ca2+ concentration was 100 ,tM. One hundred percent of NOS activity correspond to the conversion of 7.35 pmol of arginine to citrulline per hour per mg. (Inset) Activation of NOS activity as a function of CaM, and potentiation by NCS-1 of CaM-stimulated NOS activity. NOS activity was assayed using various CaM concentrations (1-300 nM) in the absence (lower trace) or in the presence (upper trace) of NCS-1 (1 ,uM). NCS-1 was able to potentiate CaM-dependent activity (P < 0.001) at each CaM concentration tested. Downloaded by guest on September 30, 2021 9256 Neurobiology: Schaad et al. Proc. Natl. Acad. Sci. USA 93 (1996)

activity (Fig. 2C Inset). This potentiation level was observed following exactly the same procedure used to purify NCS-1 (8). throughout the entire range of CaM concentrations tested. In The two additional affinity-purified proteins are shown in Fig. addition, we noted that at a very low (10-9 M) CaM concen- 3B, and their activity was first revealed in a rhodopsin phos- tration, NOS activity was markedly enhanced by NCS-1 at a phorylation inhibition assay (8). However, unlike NCS-1 in the higher level than with supramaximal concentrations of CaM PDE assay, neither Recoverin nor Vilipl could directly acti- alone. This observation suggests that the combination of vate PDE (Fig. 3A). Although the homology of CaM for Vilipl NCS-1/CaM is 2-4 times more effective than CaM alone in (22% of identity) or Recoverin (26%) are similar to the stimulating NOS activity. homology between CaM/NCS-1 (21%), only NCS-1 seems The Specificity of the NCS-1 Effect. To ensure that the able to activate PDE in the absence of calmodulin. In addition, NCS-1 activity reported above was due to NCS-1 itself and not the protein preparation responsible for the NCS-1-dependent to a bacterial protein that copurifies on the affinity column, we activation of PDE, calcineurin, and NOS can form crystals overexpressed two other proteins of the NCS family (Recov- (shown in Fig. 3C) under several experimental conditions. erin and Vilipl) in E. coli and purified them individually by After extensive washing, dissolution, and analysis by SDS/ PAGE (Fig. 3D), these crystals are found to contain a protein with a relative molecular weight (-22 kDa) identical to NCS-1. A B. Taken together, these results indicate that NCS-1 itself is responsible for the activation of the three calcium-dependent

G cr enzymes described above. kDa Intracellular Concentration ofCaM Versus NCS-1. A West- cE 1-106 0 I-65 ern blot estimate for the in vivo concentration of NCS-1 i-49.5 indicated that chicken retina or brain extracts contain -0.5-5 0 the concentration !-325 ,uM ofnative NCS-1 (Fig. 4). By comparison, D 1-275 of CaM in the brain is higher, at -3-12 10-5 M (33, 34). LI; However, although NCS-1 is 10- to 100-fold less potent than |-1B 5 CaM, it is likely that there will be an excess of NCS-1 available for its target interactions, since the concentration of CaM

o ---* .4 ...... 4- needed for the stimulation, of CaM-dependent enzymes is 0 0 1 [P.A...) ("M) thought to be -1-50 10-9 M (35) . Colocalization of NCS-1, NOS, and Calcineurin in the C D Central Nervous System. To examine whether NCS-1 was 365 coexpressed in neurons positive for NOS or calcineurin, high-

26.6- affinity antibodies against NCS-1, NOS, and calcineurin were :. ...j -NCS-l used to compare the cellular localization of these three en- 20O O-_A:d.i3l, zymes in the nervous system. We found several brain areas

14 3- where NCS-1 and NOS/calcineurin could potentially interact. In rat brain, NCS-1 and calcineurin were coexpressed in a 65 - i.. ! variety of easily recognized cell types-e.g., hippocampal CAl pyramidal neurons (Fig. 5 A and B)-or striatal medium spiny 34 - neurons (data not shown). Since PDE is also prominent in hippocampal pyramidal neurons (36), it is very likely that FIG. 3. Specificity of the NCS-1-dependent effect. (A) Activation NCS-1 could interact with calcineurin and PDE in this neuron of PDE activity as a function of NCS-1, Recoverin, and Vilipl. The dose-response curves were determined as described in the legend of population. Coexpression between NOS and NCS-1 was best Fig. LA using various concentrations of three NCS proteins (-, NCS-1; observed in the cerebellar basket cell axons (Fig. 5 C and D). 0, Recoverin; A, Vilipl), each overproduced in E. coli using the pET These cells have axons which ramify around the vector system (32) and affinity-purified individually using the same body and initial segment and form a characteristic structure, protocol as described (8). Only NCS-1 was able to increase and saturate PDE activity. One hundred percent of PDE activity (1-fold stimulation) corresponds to the hydrolysis of 0.01 ,umol of 3':5'-cAMP z w m to 5' AMP per min per mg. (B) SDS/PAGE of the NCS proteins used in the PDE assay shown in A. Five micrograms of each NCS protein or of CaM (Sigma) was analyzed on a 13% SDS polyacrylamide gel and kDa revealed by Coomassie blue staining. Bands corresponding to NCS-1 _ lIiR,70 (21,879 Da), Recoverin (23,335 Da), Vilipl (22,144 Da) or CaM _ 11 > iiiii g 106 (17,800 Da) are visible. Note that the three NCS proteins tend to form 80 dimers (43.6-46.6 kDa) in these running conditions. The size in kDa of the molecular weight markers are indicated. (C) Crystallization of NCS-1 using sparse matrix screen conditions. NCS-1 crystals were grown at 4°C using the hanging drop technique. Aliquots of protein (25 ..$.$!'I.$$.-.! ..g.s .....32 mg/ml initial protein concentration) were combined with equal ali- 11...... 27 quot of precipitant solution [2% (wt/vol) PEG 8000, 1 M LiSO4- (H20)4, and equilibrated against excess precipitant solution. Dimen- sion of largest single crystals were estimated at 200 ,um x 100 ,um x 50 ,um. (D) Authentication of NCS-1 crystals by SDS/PAGE. Crystals of NCS-1 grown using conditions described in C were extensively washed with mother liquor, dissolved in electrophoretic loading buffer, and analyzed on a 15% SDS polyacrylamide gel (right lane). Protein detection was carried out by silver staining. Proteins used for relative FIG. 4. Estimate of the NCS-1 quantity in the nervous system by molecular weight markers (left lane) were lactate dehydrogenease Western blot analysis. Purified recombinant NCS-1 (4 ng) or native (36.5 kDa), triosephosphate (26.6 kDa), trypsin inhibitor NCS-1 (-22 kDa) present in chicken brain and eye extracts were (20.0 kDa), lysozyme (14.3 kDa), aprotinin (6.5 kDa), and insulin B visualized by autoradiography with affinity purified polyclonal NCS-1 chain (3.4 kDa). Relative molecular weight of NCS-1 was estimated at antibodies. The amount of native NCS-1 was estimated at -0.5-5 ,M. 22 kDa. The size (in kDa) of the molecular weight markers are indicated. Downloaded by guest on September 30, 2021 Neurobiology: Schaad et aL. Proc. Natl. Acad. Sci. USA 93 (1996) 9257 A B 3D

50 50

E 2

35 i 40 \ 12503 35 5fi _

10 15..~~~~~~~~~~~~125; ,jr35

xuU 21t/~~~~~~~~~~~~~~~~~025 4 30 0 10 20 30 40 Ume after Injecton (hr) INCS-1] (pM) FIG. 6. The in vivo effect of NCS-1. (A) Effect of NCS-1 on motile behavior of live Paramecium. Wild-type Paramecium calmodulin, or NCS-1, or a control buffer was injected into 15 cells (n = 4) of i.l~~~~~~~~~~H Paramecium calmodulin mutant called cam1 as described (19). The 1M,: graph gives the backward swim time that resulted from cam] injected cells placed in a strongly stimulating Na+/tetraethyl ammonium chloride solution that causes backward swimming in wild-type cells of -5-10 sec (19). NCS-1 was able to partially restore the cam1 mutant phenotype to wild type. The NCS-1 effect was first detected 4 h after 9t. the injections, reached a maximal level after 12 h (not shown), and was not observed "30 h after the injections. (B) Restoration of wild-type Paramecium behavior as a function of NCS-1 concentration. cam1 mutant cells were injected as described above with 10 pl of a solution containing various amount of NCS-1 (16-160 pg). Assuming a cell FIG. 5. Immunolocalization of NCS-1 and calcineurin in hip- volume of about 200 pl, the final concentration of NCS-1 ranged from pocampus and of NCS-1 and NOS in the cerebellum of adult rat brain. 4 to 40 ,uM. The in vivo effect of NCS-1 on cam1 was saturable (EC5o (A and B) Localization of NCS-1 (A) and calcineurin (B) in the = 5 ,tM; n = 3). hippocampal pyramidal cell layer in area CA-1. Both proteins were localized within pyramidal cell bodies and dendrites. (C and D) whereas injections with native CaM prepared from Parame- Localization of NCS-1 (C) and NOS (D) in cerebellum. Both proteins cium produced faster and higher responses. were found within several cell types within the cerebellum, but the most striking colocalization occurred within the ramifications of DISCUSSION basket cell axons, including the characteristic pinceau (arrows) formed by the basket cell axon around the Purkinje cell axon initial segment. The mechanism underlying the stimulation by NCS-1 is not pcl, Pyramidal cell layer; PC, Purkinje cell. (Bar = 20 gm.) known, but it could be similar to the control mechanism for the pinceau (arrows Fig. 5 C and D). Both NCS-1 and NOS CaM-regulated enzymes or ion channels. In the case of to stain the in the cerebellar cell calcineurin or PDE, an auto-inhibitory domain of the enzyme appeared glomeruli granule unless binds to an over- layer very intensively (data not shown). Since NOS occurs also prevents catalytic activity Ca2+-CaM in numerous neuronal NCS-1 and NOS must lapping domain that then removes the influence of the inhib- populations, domain Evidence for such a interact in several other areas of the nervous system. Finally, itory (42, 43). disinhibitory coexpression and colocalization of CaM and NCS-1 in the mechanism by Ca2+/CaM has also been proposed for the central nervous is to occur since CaM is modulation of olfactory (3) and photoreceptor (2) cyclic system likely present nucleotide and for the activation of the in neurons (37-39). gated channels, Partial Restoration of CaM-Defective Mutants by NCS-1. Ca2+-dependent K+ channels in Paramecia (40). It is likely, numerous in vitro the role of CaM in vivo and although not yet proven, that a similar modulatory mechanism Despite studies, is used to restore the mechanisms by which CaM coordinates its numerous by Ca2+/NCS-1 the activity of the Ca2+- targets in living cells are still unclear. CaM directly modulates dependent K+ channels in cam1 Paramecium and to activate several types of ion channels both in neurons and in the PDE or calcineurin. By contrast, the mechanism underlying unicellular organism Paramecium tetraurelia (40). Mutations in the potentiation of NOS activity by NCS-1 remains to be the single-copy CaM gene of Paramecium were obtained from identified, since the presence of CaM is required to observe an a class of live mutants (caml) which tend to over-react to effect of NCS-1. Our data suggest that neurons expressing both various stimuli by a longer backward swim time (avoiding NCS-1 and CaM have theoretically a greater capacity to reaction) (41). Electrophysiological data revealed that cam] synthesize NO in response to internal calcium concentration mutants have lost their Ca2+-dependent K+ current (19). ([Ca2+]i) increases. To evaluate the dual role of CaM and Injection of wild-type CaM into cam1 mutants restores the NCS-1 in the activation of their common target enzymes, the normal behavioral response through cytoplasmic complemen- relative amount and activity of CaM versus NCS-1 in neurons tation. The restoration of the wild-type phenotype lasts for must be taken into consideration. A neuron may have a low "20-30 h before returning to the mutant phenotype (19). To level of CaM and a high concentration of NCS-1 or vice versa. test whether or not NCS-1 could replace CaM in vivo, we The subcellular localization of CaM and NCS-1 could also be injected highly purified NCS-1 into cam' mutants. We mea- very different for these two calcium binding proteins. sured the backward swim time of injected cells placed in a Controls experiments with extracts from bacteria not ex- Na+/tetraethyl ammonium solution which causes backward pressing NCS-1 or expressing other NCS-related proteins did swimming of -5-10 sec in wild-type cells and of '40-50 sec not have any effect in the various assays. Moreover, the NCS-1 in cam] mutants. We observed that NCS-1 alone was able to preparation used in this study produces crystals, and, there- partially restore the cam1 mutant phenotype to wild type (Fig. fore, rules out a possible contamination with a calmodulin-like 6A). As shown in Fig. 6B, the half-maximal activation (EC5o) activity. The EC5o values indicate that NCS-1 is not as good as was observed with '5 ,uM of NCS-1. This effect was first CaM for stimulating PDE, calcineurin, or NOS activity, al- observed 4 h or 12 h (data not shown) after the injections. though the NCS-1-dependent effect is saturable, and specific. Control injections with a simple buffer gave no responses, This may indicate that the in vivo target of NCS-1 is still Downloaded by guest on September 30, 2021 9258 Neurobiology: Schaad et al. Proc. Natl. Acad. Sci. USA 93 (1996) missing. Alternatively, the lower affinity of NCS-1 for its 13. Jancarik, J. & Kim, S. H. (1991) J. Appl. Crystallogr. 24,409-411. targets may be compensated by its higher affinity for Ca2 . 14. Kincaid, R. L. & Manganiello, V. C. (1988) Methods Enzymol. Indeed, the NCS-1 sensitivity for Ca2+ is so high, that a 159, 457-470. transient change in the concentration of free Ca2+ in the cell 15. Sikkink, R., Haddy, A., MacKelvie, S., Mertz, P., Litwiller, R. & to 1.5 ,uM will produce a shift from 10% to 90% Rusnak, F. 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