Center for Research on Occupational and Environmental Toxicology (C.N., M.I., H.G., M.R., N.F., C.A.), Departments of Physiology and Pharmacology and Behavioral Neuroscience (K.N.), Oregon Health Sciences University, Portland, Oregon 97201; Medical Research Service (K.N.), Veterans Affairs Medical Center, Portland, Oregon 97201; Advanced Research Institute for Science and Engineering (M.I.), Waseda University, Tokyo 169, Japan; and Department of Molecular Neurobiology (T.Y.), School of Human Sciences, Waseda University, Tokorozawa 359, Japan
Address all correspondence and requests for reprints to: Charles N. Allen, CROET L606, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97201. E-mail: allenc{at}ohsu.edu
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ABSTRACT |
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INTRODUCTION |
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The melatonin 1a receptor is a seven-transmembrane domain
(7TM) receptor (5) that signals intracellularly via G
proteins (16). Melatonin receptors regulate a wide range
of pertussis toxin (Ptx)-sensitive intracellular messenger pathways
(17, 18, 19). These activities include inhibition of forskolin
(Fsk)-stimulated cAMP production (5, 20), potentiation of
Gs-stimulated adenylyl cyclase activity (21), long-term
sensitization of Fsk-stimulated cAMP production (22), and
potentiation of PGF2-activated PLC (19). Melatonin
receptors also couple to Ptx-insensitive Gz to control cAMP
(23) and Gq to control intracellular calcium
(24). Coupling to Gq may also mediate activation of PKC
(25) by melatonin. In addition to intracellular second
messengers, melatonin receptors activate Kir3 (GIRK) inward rectifier
potassium channels (15). Kir3 channels are widely
expressed in the brain including the SCN (26, 27) and
pituitary (26) and are directly activated by
membrane-bound ß
-subunits (28) released by
Gi/Go-proteins. In rat pituitary gonadotrophs, melatonin modulates
calcium channels (7), which can also be directly inhibited
by ß
- subunits (29). Thus, melatonin receptor
regulation of these ion channel types through G proteins is important
in understanding the activities of melatonin in different tissues
in vivo.
Recently, a G (Gly) to T (Thr) point mutation was identified in TMVI of
the human melatonin 1a receptor, which controls melatonin binding
(30). This demonstrated that, as for other monoamine
receptors, binding of the cognate ligand occurs within the receptor
cleft formed by the 7TM domains. However, receptor sequences that
control signaling through G proteins have not been identified.
Cytoplasmic domain II of 7TM domain receptors lies between TMIII and
TMIV. This region has been studied in several biogenic amine and
peptide receptors and shown to be involved in signal transduction
through G proteins (31). An amino acid sequence motif
D/ERY/WXXI/VXXPL has been described near the end of TMIII
(32). Mutation of the highly conserved acidic residues (D,
Asp or E, Glu) found in virtually all 7TM receptors has caused effects
ranging from loss of function (33, 34) to constitutive
activity (35, 36, 37, 38, 39). For instance, 2A-adrenergic
receptors mutated from DRY to NRY in this position are unable to
inhibit cAMP (33). Because melatonin receptors are unusual
in possessing the neutral amino acid N (Asn) in this position, point
mutations were created in the human melatonin 1a (mt1) receptor
including consensus reversions to D/ERY at position N124. The effects
of these mutations with respect to receptor expression, ligand binding
and signal transduction to cAMP production, potassium channels, and
intracellular calcium fluxes were then examined in a stably transfected
neurohormonal cell line. The unusual transition from aspartate (D) to
asparagine (N) at this position within the melatonin receptor is not a
conservative change but a meaningful divergence from the majority of
other 7TM receptors. This amino acid has functional control of cAMP
levels and ion channel activity and a unique effect on melatonin
binding and receptor distribution within the cell.
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RESULTS |
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Inhibition of cAMP Production
Melatonin inhibition of Fsk-stimulated cAMP production was
concentration dependent in N124N (wt) expressing cells
(IC50 = 0.88 nM; Fig. 2A). Melatonin did not inhibit
Fsk-stimulated cAMP production in the parent AtT20 cells (2 ± 2%
inhibition, data not shown). The concentration dependence for
inhibition of cAMP production was affected by N124D
(IC50 = 4.4 nM, P =
0.03; Fig. 2B
) but not N124E (IC50 = 2.2
nM, P = 0.64; Fig. 2C
) point
mutations. Melatonin efficacy was significantly decreased for N124D
(27.5 ± 2.4% inhibition at 1 µM,
P < 0.001) and N124E (16.6 ± 1.9% inhibition,
P < 0.001) compared with wild-type N124N (40.9 ±
1.6% inhibition). Several independent cell lines were tested for
melatonin inhibition of Fsk-stimulated cAMP using a single
concentration of melatonin (1 µM, Fig. 2D
).
Melatonin inhibition of cAMP production in N124N (wt) cell lines
(47 ± 3%, Fig. 2D
) was significantly greater than in N124D
(27 ± 4%, P < 0.002) and N124E (20 ± 4%,
P < 0.0003) cell lines. Inhibition of cAMP was not
significantly different when comparing N124D and N124E cell lines as
groups (P > 0.267). Therefore, the N124D and E
receptors were functionally deficient, indicating impaired
intracellular signaling by the receptors.
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Potassium Channel Activation
Melatonin receptors have been shown to couple to Kir3.1/3.2
channels in vitro by a Ptx-sensitive mechanism
(15). The subunit composition of Kir3 channels in AtT20
cells was determined using RT-PCR on total RNA isolated from the
wild-type AtT20 parent cells. PCR products were Southern blotted by
probing with subunit specific
32P-oligonucleotides, demonstrating expression of
Kir3.1 (GIRK1) and Kir3.2 (GIRK2) subunits but not Kir3.4 (GIRK4)
subunits in AtT20 cells (Fig. 3A). A
similar result was obtained by Northern blot analysis of AtT20
poly-A+ RNA (data not shown). Thus, AtT20 cells
express Kir3.1/3.2 channels, the predominant subunit composition found
in the brain (27).
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Inhibition of Intracellular Ca++ Fluxes
Melatonin receptors inhibit voltage-dependent calcium channels
in vivo (41). AtT20 cells express several
voltage-dependent calcium channel types including L-, N- and
P/Q-channels (42). These channels can be activated by
depolarization of the cell membrane with high external
K+ (HiK, 30 mM; Ref.
29). HiK application to AtT20 cells induced an increase in
intracellular calcium measured by Fura-2 fluorescence ratio imaging
that was approximately 2-fold over background (Fig. 4). Repeated application of HiK to
untransfected parent AtT20 cells caused a basal, nonspecific reduction
of K+-induced intracellular
Ca++ fluxes (13 ± 4% inhibition,
data not shown). For the N124N (wt) cell line, melatonin caused
a reversible, dose-dependent inhibition of
K+-induced calcium influx with an
EC50 = 1.2 nM (N124N =
46 ± 3% inhibition at 100 nM; Fig. 4
, A
and B). The N124D and N124E mutations strongly compromised melatonin
efficacy and potency for inhibition of K+-induced
intracellular Ca++ fluxes (N124D = 23
± 2% and N124E = 24 ± 3% inhibition at 100
nM; Fig. 4B
). This appeared to be specific to the
melatonin receptors because somatostatin (1 µM)
applied to the same cell lines reversibly inhibited
K+-induced calcium fluxes (N124D = 48
± 5%, N124E = 37 ± 7%; data not shown) similar to the
wild-type receptor (N124N = 31 ± 4%, data not shown).
Therefore, point mutation of N124 to either D or E interfered strongly
with signal transduction to voltage-dependent calcium channels.
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AtT20 cells also showed spontaneous calcium oscillations, which have
been shown to correlate with the firing of spontaneous action
potentials (43). Spontaneous intracellular calcium
oscillations recorded by Fura2 ratio imaging were found in a subset of
cells from each line (N124N, 48 ± 12%; N124D, 45 ± 12%;
N124E, 35 ± 7% spontaneously active cells) and the parent AtT20
cell line (51 ± 13% spontaneously active cells). The predominant
response to melatonin in spontaneously active N124N (wt) cells was a
reversible inhibition that was sensitive to Ptx treatment (Fig. 4, C
and D). Melatonin did not inhibit spontaneous intracellular calcium
fluxes in untransfected AtT20 cells (data not shown). Inhibition of
spontaneous calcium oscillations by melatonin was significantly reduced
in N124D and N124E cell lines (Fig. 4D
). Therefore, wild-type N124N
melatonin receptors inhibit calcium oscillations in spontaneously
active AtT20 cells by a Gi/Go-mediated mechanism. Mutation of N124 to
either D or E appeared to eliminate the melatonin control of
spontaneous calcium fluxes.
Golgi Retention of Nonfunctional Mutants
Cell lines expressing N124A, -L, and -K receptors were chosen by
intensity of staining for the N-terminal peptide tag but showed no
significant binding of 125I-melatonin (Fig. 1 and
Table 1
). In addition, N124A and N124K receptors showed a different
staining pattern compared with N124N (wt) cells: a single large
intracellular inclusion and a decreased staining intensity near the
periphery of the cells (Fig. 1C
). N124L cell lines showed staining
near the surface membrane but with a more aggregated appearance
than for N124N (wt), N124D, and N124E receptors (Fig. 1C
). In those
N124A and N124K cells that stained for
-mannosidase II, receptor
staining showed a strong colocalization (Fig. 5A
), indicating retention of the receptor
proteins in the Golgi (44). Receptor staining was also
observed which did not colocalize with
-mannosidase II, indicating
presence of the additional receptors in a separate compartment (Fig. 5B
). Thus, amino acid position N124 had far-reaching effects on the
structure and conformation of the melatonin receptor. In addition to
functional effects on melatonin potency and efficacy seen with N124D
and N124E mutations, other substitutions at this position caused a loss
of agonist binding and retention of the receptor in the Golgi. This
indicates that N124 controls not only receptor signaling function but
is critical for correct receptor structure.
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DISCUSSION |
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Within the D/ERY motif, the second position R (Arg) residue has
been modeled to rotate in and out of the agonist binding cleft and
serve as an agonist-sensitive conformational switch controlling G
protein interaction (36, 45). This portion of the receptor
is proximal to the cell membrane and therefore at an interface between
hydrophilic and hydrophobic regions. Thus, the charge character of the
residue adjoining the conserved R residue in the D/N position could
also influence receptor movement or interaction with G proteins.
Substitution of the neutral N (Asn) with nonpolar, hydrophobic L (Leu),
A (Ala), or positively charged K (Lys) eliminated agonist binding
(Table 1) and affected the subcellular distribution of receptor protein
(Fig. 1
). N124A and K receptors showed a staining pattern with a single
large intracellular inclusion associated with the medial or trans-Golgi
(Fig. 5
and Ref. 44) and a decreased staining intensity
near the periphery of the cells. The same characteristics that endow
functional control of signal transduction on this position through
flexibility in the receptor may also restrict the allowable amino acids
because of structural constraints. Thus, the apparent flexibility of
this part of the receptor may have consequences for assembly or folding
of the protein and explain the almost absolute conservation of the
acidic D/E amino acid residues found in most 7TM receptors.
The N124L mutant also lacked agonist binding (Table 1) but had a unique
staining pattern near the surface membrane (Fig. 1
) and thus trafficked
out of the Golgi compartments, unlike the N124A and K mutants. But,
when viewed through consecutive confocal slices, N124L had a more
aggregated appearance than N124N (wt), D, or E receptors (data not
shown). Thus, the N124L mutation may separate a conformational defect
in agonist binding from a defect related to trafficking. It is possible
that the leucine side chain, which is similar in size and structure to
asparagine, caused a less severe disruption of the receptor. These
speculations as to the folding and structural defects for N124 mutants
may be testable by associational rescue using wild-type receptor or
compensatory mutations in cotrafficking molecules to restore agonist
binding and cellular trafficking.
Receptor folding and transport can require association with cyclophilins or other chaperonins as for rhodopsin (46) and red/green opsins (47). Misfolded receptors can aggregate and fail to traffic. Alternatively, the point mutations may have interfered with an associated subunit required for folding and trafficking. Odorant receptors have type-specific trafficking chaperones that associate with the nascent receptor in the endoplasmic reticulum (48), and these receptors fail to express in some cell lines, presumably due to the lack of expression of their required trafficking chaperone. For melatonin receptors, this seems unlikely as a cell line-specific failure of ectopic expression (49) has not yet been reported. LH receptor mutated at the homologous position, E441Q, was retained in a detergent-soluble compartment that could not be detected with a live cell binding assay (50), indicating retention or altered internalization of the mutant receptor. This is of interest as the amino acid glutamine (Q) is of similar structure and neutral charge as asparagine (N). Thus, an analogous, but inverse transition may have resulted in a similar phenotype; however, intracellular localization of the mutant receptor by fluorescent staining or a similar method was not examined.
Identification of the functional determinants that control second
messenger signaling is important for understanding the actions of
melatonin in different tissues. Melatonin receptors signal to a broad
range of intracellular effectors. Acidic substitutions for N124 had
distinct effects on each of the three signaling pathways that were
assessed. The maximal inhibition of cAMP production by melatonin was
decreased but still detectable (Fig. 2), whereas Ptx-sensitive
inhibition of calcium influx was abolished (Fig. 4
) and activation of
Kir3 potassium channels was unchanged (Fig. 3
). These results could be
explained by a mutation-induced, selective loss of coupling efficiency
by the receptors for particular combinations of G protein
ß
-subunits. The functional outcome of this selective loss of
coupling would then depend on the G protein specificity of the
effector. For example, muscarinic and somatostatin receptors have been
shown to prefer specific
ß
combinations in coupling to calcium
channels (57). In addition, N-type calcium channels are
selective for ß-subunits, binding strongly to ß1 and ß2 and
weakly to ß3, ß4, and ß5 (51). A selective loss of
coupling by N124D and N124E receptors to Ptx-sensitive Gß1- and
Gß2-containing heterotrimers might result in the observed complete
loss of Ptx-sensitive and sparing of Ptx-insensitive inhibition of
calcium channels. On the other hand, Kir3 (GIRK) potassium channels
have no specificity for subtypes, except for not responding to ß5
(52). Thus, activation of Kir3 channels would not be
affected by a mutation-induced loss of coupling to Gß1- and
Gß2-containing heterotrimers. Alternatively, the relatively small
K+ currents evoked in AtT20 cells may have
limited the ability to resolve differences in signaling strength.
Adenylyl cyclases are not known to show a preference for inhibition by
particular subtypes of G
i or Gß
(53), so partial loss of inhibition of adenylyl cyclase is
consistent with a selective loss of mutant receptor coupling to a
subset of the total pool of heterotrimers that are activated by the
wild-type receptor.
Although all three pathways were sensitive to inhibition by Ptx
treatment, indicating the involvement of the Ptx-sensitive G proteins
Gi and Go, there was a Ptx-insensitive component of the regulation of
K+-activated calcium fluxes by the melatonin
receptor (Fig. 4). Similarly,
2-adrenergic receptors have been shown
to inhibit calcium channels by both Ptx-sensitive and -insensitive G
proteins and, as observed for melatonin receptors, the
IC50 for agonist was significantly higher for
Ptx-insensitive inhibition (54). This could reflect
coupling to another G protein pathway. Indeed, a secondary molecular
interaction between melatonin 1a receptors and Gq has recently been
demonstrated (24).
For 7TM receptors signaling to phospholipases through Gq proteins, the
transition from D to N at a position homologous to N124 appears to be
an essentially conservative substitution. 1B-adrenergic
receptor mutation at D142 resulted in constitutive inositol
phosphate accumulation for most of the substitutions tested
(36). However, D142N and E showed no major effect on
agonist affinity and only small effects on constitutive inositol
phosphate accumulation (35, 36). A similar result was
found for the Gq-coupled m1 muscarinic (D122N, Ref. 37),
m5 muscarinic (D127N, Ref. 38), and 5HT2 (D172N, Ref.
39) receptors where the homologous, inverse mutations had
little effect on agonist-stimulated or constitutive activities. Thus,
it is reasonable to expect a disruption of Gi but not Gq signaling by a
mutation at N124 in melatonin receptors, but this remains to be
tested.
The "D/ERY" motif found in virtually all other 7TM receptors has been shown to affect signal transduction through Gi, Gq, and Gs depending on the receptor examined. The effect of this type of substitution in "D/ERY" receptors or other receptors possessing the neutral amino acid N (Asn), which includes the platelet-activating factor receptor (55), tagged so that their trafficking can be examined remains to be tested. Whether due to the physical requirements of melatonin as a ligand, the required G protein specificity for proper melatonin signaling, or another structural requirement related to receptor synthesis, folding, or transport, position N124 clearly differentiates the melatonin receptor from most 7TM receptors.
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MATERIALS AND METHODS |
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Point mutations were generated using oligonucleotide-directed mutagenesis as described (Transformer Site-Directed Mutagenesis, CLONTECH Laboratories, Inc. Palo Alto, CA) using the following oligonucleotides (Life Technologies, Inc., Gaithersburg, MD): 5'-GTG ACT GGT GAA TAC TCA ACC AAG-3' (selection marker), 5'-GGC ATC GCC ATC GAC CGC TAC TGC-3' (N124D), 5'-GGC ATC GCC ATC GAG CGC TAC TGC-3' (N124E), 5'-GGC ATC GCC ATC GCC CGC TAC TGC-3' (N124A), 5'-GGC ATC GCC ATC CTC CGC TAC TGC-3' (N124L), 5'-GGC ATC GCC ATC AAG CGC TAC TGC-3' (N124K). Each cDNA was then completely sequenced by automated sequencing (Stretch 373, Perkin-Elmer Cetus, Norwalk, CT) and compared with the published receptor sequence using the Factura/Autoassembler (Perkin-Elmer Cetus) and GCG suite of programs (Accelrys, Inc., San Diego, CA) to confirm the presence of a single, exclusive mutation site.
RT-PCR
RT-PCR was performed by extraction of total RNA from culture
cells or brain tissue prepared by a modified guanidinium thiocyanate
extraction (56). RNA (15 µg) was primed using
oligo-d(T)18 and reverse transcribed with
Superscript-RT (Life Technologies, Inc.) under standard
reaction conditions and used as template for PCRs performed using
Taq polymerase (Promega Corp., Madison, WI) in
a Perkin-Elmer Cetus 9600 PCR machine in standard reaction
buffer. Sequences of the oligonucleotide used for Kir3 channel RT-PCR
and Southern blot were: 3.15', 5'-AACAGCCACATGGTCTC-3'; 3.13',
5'-GTGCTAATGTCATCTAGTC-3'; 3.1-probe,
5'-TTGACCAACTTGAACTGGATGTAGGTTTTAGTACAGG-3'; 3.25',
5'-TCAACGCCTTCATGGTA-3'; 3.23', 5'-CTTGATCCACACTAGGA-3'; 3.2-probe,
5'-CTCCAAAGCGCAGCTGCCTAAAGAGGAACTGGAGA-3'; 3.45',
5'-GTATGGCTTCAGAGTCATT-3'; 3.43', 5'-TTCATCCTTCTCGGCCT-3';
3.4-probe, 5'-CTTTTGTGGAGATGTCTCGTGCTCAACTGGAACAG-3'; cyclophilin-5',
5'-CTGGTCTTGCCATTCCTGGACCCA-3'; cyclophilin-3',
5'-CATGTGCCAGGGTGGTGACTTCAC-3'. The conditions for Kir3 PCR cycling
reactions were: 94 C/30 sec; 58 C/30 sec; 72 C/60 sec for 35 cycles.
The conditions for cyclophilin PCR cycling reactions were: 94 C/60 sec;
60 C/30 sec for 35 cycles.
Cell Culture and Transfection
Mouse anterior pituitary tumor cells (AtT-20/D168) cells are
grown in DMEM (4,500 mg/liter glucose, Life Technologies, Inc.) plus 10% FBS (Life Technologies, Inc.) at 37
C and 5% CO2. For stable transfections cells
were trypsinized and plated (5 x 104
cells/ml, 0.3 ml/cm2) and at 1 d after
plating were transfected using Lipofectamine Plus (Life Technologies, Inc., 0.1 ml/cm2, 1 µg/ml
plasmid, 3 µl/ml Lipofectamine Plus, 1.5 µl/ml
lipofectamine) for 418 h before media change with media changes every
24 h thereafter. Cells were selected using blasticidin
S.HCl (2.5 µg/ml, Invitrogen)
starting at 2 d after transfection, reapplied with media daily for
10 d and every 2 d subsequently. At 1014 d after
transfection, cells were scraped and plated at high dilution and single
colonies were picked to establish cell lines. For routine maintenance,
blasticidin and media were renewed every 4 d. For functional
assays blasticidin and media were changed every 2448 h and always
applied the day before assay.
125I-Melatonin Binding
AtT20 cells were harvested by scraping in PBS, pH 7.4, pelleted,
and resuspended in approximately 10 ml lysis buffer at 4 C containing
(mM): 50 Tris-HCl, 1 PMSF, and 1 EGTA at pH 8 and
homogenized (Brinkmann Instruments, Inc., Westbury, NY).
Membranes were then pelleted at 36,000 x g for 20 min
and resuspended in sodium ion-free assay buffer at 25 C containing 50
mM Tris-HCl and 5 mM
MgCl2 at pH 7.4.
2-[125I]-melatonin was incubated with membranes
(30 µg total protein/500 µl) in the absence and presence of
melatonin for 150 min at 25 C with continuous shaking. The reactions
were terminated by filtering over GF/B filters (Whatman,
Clifton, NJ) using a 96-well harvester (Tomtec, Orange, CT) at 4 C and
washed with pH 7.4 buffer at 4 C containing 10 mM
Tris-HCl, 150 mM NaCl. The filters were dried,
and each sample was saturated with 50 µl of scintillation fluid and
counted using a BetaPlate scintillation counter (Wallac, Inc., Gaithersburg, MD). Data for saturation and competition
binding were analyzed by nonlinear regression using commercial software
(Kaleidagraph) according to the formula: y =
Bmax * x/(x + Kd) where
y = concentration of bound 125I-melatonin
and x = concentration of free
125I-melatonin.
Immunofluorescent Imaging
AtT20 cells lines were trypsinized and plated in four-well
permanox plastic chamber slides (Nalge Nunc International,
Rochester, NY) for 24 d with daily media and antibiotic replacement.
Cells were fixed in 3% paraformaldehyde in PBS (0.58 M
Na2HPO4, 0.17 M
NaH2PO4, pH 7.4; 0.68
M NaCl) at 4 C for 30 min, washed three times with TBS-GS
(50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5.5%
vol/vol goat serum; Life Technologies, Inc.) for 5 min and
preblocked with TBS-GS for 1 h at room temperature. Cells were
then incubated overnight with primary antibody (anti-Xpress monoclonal
IgG1, Invitrogen, 1:100 dilution; rabbit
anti--mannosidase-II serum, Complex Carbohydrate Research Center,
University of Georgia, 1:500 dilution) in TBS-GS and washed three times
with TBS-GS. Cells were then incubated for 1 h with secondary
antibody (goat antimouse IgG-Alexa 488, goat antirabbit Alexa 568;
Molecular Probes, Inc., Eugene, OR) at a 1:800 dilution
and washed three times with TBS-GS. Chambers were then removed, slides
dipped briefly in distilled water, cover slips mounted with Elvanol
[120 mM Tris-HCl, pH 8.5; 140 mM 1,
4-diazobicyclo-[2.2.2]-octane; 12% (wt/vol) polyvinylalcohol; 50%
(vol/vol) glycerol] and sealed with nail polish.
Confocal imaging was performed at the Oregon Hearing Research Center with an MRC 1024 ES laser scanning confocal imaging system (Bio-Rad Laboratories, Inc., Hercules, CA) with krypton/argon laser excitation at 488 nm attached to an inverted Eclipse TE300 microscope (Nikon, Melville, NY). Images were acquired and processed using LaserSharp (Bio-Rad Laboratories, Inc.) and Photoshop (Adobe Systems, San Jose, CA) software packages. Conventional immunofluorescent images were acquired using a DMIRBE microscope equipped with high numerical oil lenses (63 x 1.32 N.A.; Leica Corp., Deerfield, IL), green fluorescent protein/ENDOW and rhodamine N3 filter sets (Chroma Technology, Brattleboro, VT), a Micromax camera with Sony chip (1300 x 100 pixels; Princeton Instruments, Monmouth Junction, NJ) and Metamorph ver. 4.1 software (Universal Imaging Corp., Westchester, PA).
cAMP Production
For cAMP assay, cells were trypsinized, plated onto 24-well
plates (Falcon, Franklin Lakes, NJ), and changed to fresh
culture media with antibiotic every 48 h thereafter. At three to
five days after passage, 8090% confluent cells were rinsed three
times in DMEM, incubated with 1 mM
3-isobutyl-1-methylxanthine (RBI, Natick, MA), 500
µM 4-(3-butoxy-4-methoxy-benzyl)imidazolidin-2-one
(Sigma, St. Louis, MO), and agonist for 10 min before the
addition of 100 µM Fsk. After an additional
incubation of 20 min, the medium was aspirated, 0.5 ml of 4
mM EDTA, 10 mM Tris-HCl, pH 7.5, were added on
ice, and the plates were frozen at -80 C before assay. For assay each
sample was harvested, sonicated, boiled for 5 min, placed on ice, and
then centrifuged (12,800 x g for 20 min). Each
supernatant (50 µl) was assayed for cAMP levels in duplicate
according to standard methods (3H-cAMP assay no.
TRK432, Amersham Pharmacia Biotech, Arlington Heights,
IL). Fsk stimulation typically resulted in 10- to 12-fold cAMP produced
over background levels (4 pmol cAMP per well) in the presence of
3-isobutyl-1-methylxanthine/4-(3-butoxy-4-methoxy-benzyl)imidazolidin-2-one.
Results were expressed as a percentage of Fsk-stimulated maximum except
for responses using Ptx-treated cells. Ptx treatment caused a
nonspecific increase in Fsk-stimulated cAMP levels in all wild-type and
melatonin receptor stable cell lines. For Ptx treatment, results were
calculated as a percentage of a combined maximum stimulation by both
Fsk and Ptx. Curve fits were determined using the logistic growth model
(57), which has the functional form: f(x | m0, m1,
m2) = y = m0 + m1/(1 + m2e-[x]) where m0 and
m1 are minimum and maximum response values calculated for individual
response values y at each agonist concentration [x] using nonlinear
least squares algorithm provided in the R package (58).
Degrees of freedom for the statistical test were calculated by
Satterthwaite approximation (59). SE
values for the estimated IC50 and
EC50 values were found using the delta method
(59, 60).
Electrophysiology
For electrophysiological recording the cells were trypsinized,
plated at a density of approximately 5 x
104 cells/ml onto washed 18-mm glass coverslips
(no. 12545-84, Fisher Scientific, Pittsburgh, PA) and
changed to fresh culture media with antibiotic each day thereafter.
Recordings were performed at 34 d by placing a coverslip fragment
into a recording chamber located on the stage of an inverted microscope
equipped with phase-contrast optics. The chamber was perfused at
approximately 1 ml/min with extracellular solution, at pH 7.3 and 320
mOsm containing in mM: NaCl, 128; KCl, 30;
CaCl2, 3; MgCl2, 2;
glucose, 10; and HEPES, 10. Microelectrodes, pulled in two stages
(Narishige puller) from thin-walled borosilicate glass (World Precision
Instruments, Sarasota, FL), had resistances of 57 M
when filled with a (intracellular) solution, at pH 7.3 and 310 mOsm,
consisting of in mM: K+ gluconate,
84; KCl, 38; KOH, 33; CaCl2, 1; HEPES, 10; EGTA,
10; ATP, 3; and GTP, 0.3. Test agents were applied from a U-tube
system, while antagonists were applied both through the U-tube and by
bath perfusion. Compounds were made fresh before each experiment.
Currents were recorded using the whole-cell configuration of the
patch-clamp technique and voltage-clamped using an EPC-7 (Adams List),
filtered at 2 kHz (-3 dB) with an eight-pole Bessel filter and
digitized on-line using an ITC16 A/D interface (Instrutech, Port
Washington, NY) and Pulse software (HEKA, Lambrecht, Germany).
Curve fits and values for IC50 and
EC50 values were determined by nonlinear
regression using commercial software (Kaleidagraph 3.0.4) according to
the logistic formula: y = c/[1 + exp(H *
ln([C50]/[x]))] + b where b and c are
minimum and maximum response values, H is a Hill coefficient, and
[C50] is the 50% concentration calculated by
nonlinear regression for individual response values y at each agonist
concentration [x].
Calcium Imaging
AtT20 cell lines were trypsinized and plated in uncoated glass
bottom petri dishes (MatTek Corp., Ashland, MA) at a density of
approximately 5 x 104 cells/ml, media and
blasticidin were changed each day, and cells were assayed at 3 to
4 d. The cells were gently rinsed with buffered salt solution
(BSS) consisting of (mM) 128 NaCl, 5 KCl, 2.7
CaCl2, 1.2 MgCl2, 1
Na2HPO4, 10
D-glucose, and 10 HEPES-NaOH of pH 7.4 and then incubated
in BSS supplemented with 5 µM fura-2 AM (Molecular Probes, Inc.) and 0.1% albumin for 30 min at 37 C. The cells
were rinsed in BSS, incubated at 37 C for 15 min, and then continuously
perfused at a flow rate of 3 ml/min.
Fluorescent images were recorded using an upright microscope (Axioskop FS,Carl Zeiss, Thornwood, NY) with a water immersion objective (FL40x/0.8, Olympus Corp., Lake Success, NY). Paired pulse UV excitation (wavelength = 340 nm and 380 nm, each pulse length = 147 msec) was given by a monochromator (Polychrome 2, Till Photonics, Martinsried, Germany) at 6-sec intervals and reflected by a dichroic mirror (FT395 nm, Carl Zeiss). The fluorescent image was band passed (BP500530 nm, Carl Zeiss), amplified by an image intensifier (C703902, Hamamatsu Photonics, Bridgewater, NJ), and exposed to a multiple format cooled charge-coupled device camera (C4880, Hamamatsu Photonics). The UV light exposure, charge-coupled device camera, image sampling, and acquisition were controlled with a digital imaging system (ARGUS HiSCA, Hamamatsu Photonics) installed in a personal computer. All measurements were corrected for background fluorescence. Changes in intracellular concentration of Ca2+ were estimated from the ratio of fura-2 emission intensities. Curve fits and values for IC50 and EC50 values were calculated as for K+ channel measurements shown above.
Statistics
Statistical differences were calculated by unpaired Students
t test performed with commercial software (Statview 5.0.1,
SAS Institute, Cary, NC). Values were considered significantly
different when P < 0.05.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: BSS, buffered salt solution; Fsk, forskolin; HiK, high external K+; mt1, melatonin 1a receptor; Ptx, pertussis toxin; SCN, suprachiasmatic nucleus; TBS-GS, 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5.5% vol/vol goat serum; 7TM, seven-transmembrane.
Received for publication September 13, 2000. Accepted for publication May 1, 2001.
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REFERENCES |
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