From the Department of Neuroscience and Howard Hughes Medical
Institute, The Johns Hopkins University School of Medicine, Baltimore,
Maryland 21205-2185
The cGMP-gated cation channel mediating
phototransduction in retinal rods has recently been shown to be
inhibited by calcium-calmodulin, through direct binding of the latter
to the
-subunit of the heterotetrameric channel complex. Here, we
report the characterization of this inhibition and the identification
of a domain crucial for this modulation. Heterologous expression of the
- and
-subunits of the human rod channel in HEK 293 cells
produced a cGMP-gated current that was highly sensitive to
calcium-calmodulin, with half-maximal inhibition at approximately
4 nM. In biochemical and electrophysiological experiments on deletion mutants of the
-subunit, we have identified a region on its cytoplasmic N terminus that binds calmodulin and is necessary for the calmodulin-mediated inhibition of the channel. However, in gel shift assays and fluorescence emission experiments, peptides derived from this region indicated a low calmodulin affinity, with dissociation constants of approximately 3-10 µM. On
the C terminus, a region was also found to bind calmodulin, but it was likewise of low affinity, and its deletion did not abolish the calmodulin-mediated inhibition. We suggest that although the identified region on the N terminus of the
-subunit is crucial for the
calmodulin effect, other regions are likely to be involved as well. In
this respect, the rod channel appears to differ from the olfactory cyclic nucleotide-gated channel, which is also modulated by
calcium-calmodulin.
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INTRODUCTION |
Visual transduction in retinal rods involves a light-triggered
signaling cascade that leads to a decrease in cGMP concentration (for
review, see Refs. 1-3). In darkness, cytoplasmic cGMP binds to and
opens cGMP-gated, nonselective cation channels on the plasma membrane
(for review, see Refs. 4-7). These open channels sustain an inward
dark current, carried predominantly by Na+ and
Ca2+, that keeps the cell depolarized. In the light, the
hydrolysis of cGMP leads to the closure of these channels, resulting in
a membrane hyperpolarization as the electrical response. When the cGMP-activated channels close, the Ca2+ influx into the rod
outer segment stops, but a Ca2+ efflux through a
Na+/Ca2+, K+ exchanger continues,
leading to a decrease in the cytoplasmic Ca2+ concentration
in the outer segment. This decrease in Ca2+ triggers a
negative feedback to produce light adaptation of the rod (for review,
see Ref. 3). One mechanism underlying this Ca2+ feedback
involves a reduction of the apparent affinity of the channel for cGMP
(8, 9), through the action of one or more Ca2+-binding
proteins, one of which is calmodulin
(CaM)1 (10-14).
The rod channel belongs to a family of cyclic nucleotide-activated,
nonselective cation channels now known to be important for both visual
and olfactory transduction pathways (for review, see Refs. 5-7). These
channels are ligand-gated, being opened directly by cGMP and cAMP. They
are composed of at least two subunit species (
and
, or 1 and 2)
most probably forming heterotetrameric complexes (15, 16). The
-subunit, but not the
-subunit, is capable of forming functional
homomeric channels. Like the Shaker superfamily of potassium channels,
both subunits have six transmembrane domains and a putative
-hairpin
that forms part of the pore. The cyclic nucleotide-binding site is
situated on the cytoplasmic C terminus and is homologous to the binding
sites found on the cyclic nucleotide-activated kinases protein kinase C
and protein kinase A, and the Escherichia coli catabolite
gene activator protein, CAP (see Refs. 5-7).
The reduction in affinity of the rod cGMP-gated channel for cGMP
involves Ca2+-CaM binding to the
-subunit (Refs. 17 and
18; for review, see Refs. 19 and 20). A similar, but more potent,
inhibition by Ca2+-CaM was found for the olfactory cyclic
nucleotide-gated channel, although in this case the modulation involves
Ca2+-CaM binding to the
-subunit of the channel (21,
22). The mechanism by which the olfactory channel is modulated by
Ca2+-CaM has been elucidated (22). A domain on the N
terminus of the olfactory channel
-subunit influences gating by
promoting the open state of the liganded channel. When
Ca2+-CaM binds to this domain, the influence of the latter
on channel gating is removed, leading to a decrease in the apparent
affinity of the channel for cGMP due to the coupling of ligand binding and channel gating. Most recently, it has been reported that the N and
C termini of the olfactory channel
-subunit interact directly with
each other and that the domain on the N terminus that is important for
this interaction coincides with the Ca2+-CaM-binding site
(23). When Ca2+-CaM binds, the interaction between the two
termini disappears (23). Presumably, this interaction influences the
gating of the channel and accounts for the ability of
Ca2+-CaM to modulate the channel.
In this paper, we address the question of how the rod channel is
modulated by Ca2+-CaM. In an electrophysiological approach,
we recorded cGMP-activated currents in the absence and presence of
Ca2+-CaM from excised, inside-out membrane patches of HEK
293 cells expressing the human rod channel
- and
-subunits.
Furthermore, site-directed mutagenesis and biochemical binding studies
were carried out to identify the Ca2+-CaM-binding domain on
the
-subunit important for this modulation. The results indicate
similarities, but also differences, between the rod and olfactory
channels with respect to the CaM inhibition.
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EXPERIMENTAL PROCEDURES |
Cloning of the Full-length Human Rod Channel
-Subunit--
Poly(A)+ RNA was isolated from bovine
retina with the oligo(dT)-selection method (Micro-FastTrack,
Invitrogen, Carlsbad, CA). Oligo(dT)-primed cDNA was then
synthesized using the SuperScriptTM Choice system (Life
Technologies, Inc.). Using this cDNA as template, PCR was performed
with the primers AGGAAGAAGGCAAGTCCTG and ATGGGCTTGATCTCCAAGG, corresponding to nucleotides
50 to
32 and 635-617 of the cDNA coding for bovine GARP (glutamic acid-rich protein; Ref. 24). The PCR
product with the appropriate size was isolated, sequenced, and used as
a probe to screen an adult human retinal cDNA library in
gt10.
Six positive clones were isolated. The clones fell into two groups
according to their restriction patterns. One clone from each group was
used for further characterization. The inserts were subcloned into pCIS
and sequenced. These two clones, named hGARP1.6 and hGARP2.5, shared a
common 5'-region of 0.9 kb but were divergent in their 3'-regions. The
3'-region of hGARP2.5 overlapped with the 5'-end of the hRCNC2b clone
described in Chen et al. (25) (see "Results"). To
combine hGARP2.5 and hRCNC2b, two of three Bsu36I sites in hGARP2.5
were deleted by silent mutations. A
Bsu36I-BamHI fragment from hRCNC2b
(spanning nucleotides 417-2849) was then ligated into hGARP2.5,
generating a clone that appears to code for the full-length
-subunit
of the human rod channel, by comparison to the bovine ortholog. In this
paper, we refer to hRCNC2b as the trunc-
-subunit and to the
full-length
-subunit as the full-
-subunit.
Channel Protein Expression and Electrophysiological
Recordings--
The cDNAs coding for the human rod cGMP-activated
channel
-subunit (corresponding to hRCNC1 in Ref. 26 and renamed
hRCNC
here) and the trunc-
- or full-
-subunit were subcloned in
the pCIS expression vector and cotransfected into human embryonic kidney (HEK) 293 cells (American Type Culture Collection) using the
calcium-phosphate method (27). For hRCNC
, a Kozak consensus sequence
(28) had been introduced into its 5'-region, which enhanced the level
of protein expression by a modest degree. HEK 293 cells were cultured
at 37 °C in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum and penicillin/streptomycin in a humidified
atmosphere containing 5% CO2. At 2-4 days after transfection, current recordings were made from voltage-clamped, excised, inside-out membrane patches using an EPC-9 patch-clamp amplifier and the PULSE/PULSE-FIT software package (both from HEKA
Elektronik, Lamprecht, Germany, distributed by Instrutech, Great Neck,
NY). The signals were low pass filtered at 2.9 kHz (4-pole Bessel
filter). The patch pipettes were made from borosilicate glass
capillaries, with a tip-lumen diameter of 1-1.5 µm and a resistance
of 2-4 M
. The relatively low signal-to-noise ratio in some of the
traces had resulted from the small magnitudes of the currents and the
lack of signal averaging.
For zero-Ca2+ conditions, the pipette and bath solutions
both contained 140 mM NaCl, 5 mM KCl, 2 mM EGTA, and 10 mM HEPES/NaOH, pH 7.4. In
experiments involving Ca2+-CaM, the bath was perfused with
a solution containing CaM at a specified concentration and also 50 µM buffered free Ca2+ (achieved by
substituting 2 mM nitrilotriacetic acid and 704 µM CaCl2 for the EGTA). cGMP was added to the
bath solution as needed. In experiments involving the
-subunit, the
cGMP-activated current was always tested for blockage at +60 mV by 10 µM L-cis-diltiazem applied to the
bath solution to verify its functional expression (25). A
solenoid-controlled rotary valve system (29) was used to change the
bath solution, and the solution change around the membrane patch was
complete within 1-2 s. All experiments were performed at room
temperature.
Mutagenesis and Fusion Protein Construction--
Deletion
mutants were generated by performing site-directed mutagenesis on the
cDNAs coding for the trunc-
- and full-
-subunits. For binding
studies, fusion protein constructs containing the cytoplasmic N
terminus (amino acid residues 1-313) or C terminus (residues 535-908)
of the trunc-
-subunit were made by PCR amplification using primers
with flanking BamHI and EcoRI sites and
subcloning the PCR fragments into pGEX-2T (Amersham Pharmacia Biotech).
The resulting constructs were transformed into E. coli BL21
cells, and the fusion proteins were isolated and purified using the
Bulk GST purification module from Amersham Pharmacia Biotech.
Fluorescence Measurements--
Fluorescence experiments to
measure the binding of peptides to Ca2+ CaM were performed
using the Perkin-Elmer Luminescence Spectrometer LS50B. Peptides,
representing putative Ca2+-CaM-binding sites on the
trunc-
-subunit, were synthesized in the Howard Hughes Medical
Institute Biopolymer Facility at Johns Hopkins University School of
Medicine. Dansyl-CaM (Sigma) was incubated at a given concentration
with increasing peptide concentration in a buffer containing 50 mM Tris-HCl, pH 7.3, 150 mM NaCl, and 0.5 mM CaCl2 or 2 mM EGTA. The emission
spectrum at 400-600 nm was recorded using an excitation wavelength of
340 nm, the bandwidth being 10-15 nm for both excitation and emission.
The increase in fluorescence at 480 nm was used to assay for the
concentration of dansyl-CaM bound to peptide. Assuming a 1:1
stoichiometry of binding between Ca2+-CaM and peptide, the
fraction of peptide, fb, bound to CaM is given
by the equation fb = (Im
If)/(Ib
If), where If is the dansyl-CaM fluorescence with no peptide present,
Ib is the fluorescence when all dansyl-CaM is
bound to peptide, and Im is the fluorescence of
intermediate mixtures (see Ref. 22). The dissociation constant, Kd, between Ca2+-CaM and the peptide was
derived from the relationship between the fractional increase of
fluorescence and the calculated concentration of free peptide.
According to vendor specifications, the mole ratio between the dansyl
moiety and CaM in dansyl-CaM is about 0.6. However, this factor does
not influence the results, provided that CaM and dansyl-CaM behave
identically in the binding experiments.
Antibody Generation--
A polyclonal antibody, Ab1859, was
raised in rabbit (HRP Inc., Denver, PA) against the resin-coupled
peptide MLGWVQRVLPQPPGTPRKTK, which corresponds to amino acids 1-20 in
the full-
-subunit (see Fig. 1B). For immunoprecipitation
experiments, the antibody was purified by protein A-Sepharose
chromatography. Ab1859 cross-reacts with the bovine homolog of the
protein.
CaM Overlay Experiments and Western Blotting--
Overlay
experiments were performed on purified fusion proteins (see above),
retinal lysates, and the heterologously expressed full-
-subunit. Retinal lysates were prepared from human retinal tissue that had been isolated and frozen in liquid nitrogen within 5 h post-mortem. The low salt lysis buffer contained 10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 2 µg/ml
aprotinin, and 1% Triton X-100. For the heterologously expressed full-
-subunit, HEK 293 cells were harvested 3-4 days after
transfection and lysed in the above low salt buffer. The
-subunit was immunoprecipitated by incubating the cell lysate with
10 µg of the purified Ab1859 antibody and a suspension of protein
A-Sepharose (Sigma) for 2 h at 4 °C. Alternatively, the
antibody was covalently coupled to CNBr-activated Sepharose 4B and used
instead.
The fusion proteins, retinal lysates, and immunoprecipitates were
loaded on SDS gels and transferred to nitrocellulose (TransBlot, Bio-Rad) or polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford, MA) in 10 mM CAPS, pH 10.8, or Towbin buffer
containing 2-10% methanol (30). After transfer, the blots were probed
with CaM-coupled alkaline phosphatase or biotinylated CaM in the
presence of 0.1-1 mM Ca2+ or 5 mM
EGTA. The synthesis of CaM-coupled alkaline phosphatase and the
detection procedure were both according to Walker et al. (31). In assays with biotinylated CaM, the membranes were blocked in a
buffer containing 150 mM NaCl, 10 mM Tris-HCl,
pH 7.5, 1 mM CaCl2 or 5 mM EGTA,
0.1% antifoam A, and 5% nonfat dry milk for 30 min. Biotinylated CaM
(Biomedical Technologies, Stoughton, MA) was added to give a final
concentration of 1 µg/ml, followed by an incubation for 1-2 h at
room temperature. After extensive washing in the same buffer without
additives, the membrane was incubated with avidin and horseradish
peroxidase (ABC system, Vector Laboratories, Burlingame, CA) and
developed using the ECL system (Amersham Pharmacia Biotech).
For Western blotting, the membranes were blocked in 2% nonfat dry milk
in Tris-buffered saline (140 mM NaCl, 10 mM
Tris-HCl, pH 7.5) and incubated with the antibodies in the same buffer
for 1 h at room temperature or overnight at 4 °C. The antibody
Ab1859 was used at 1:2000 dilution. Fusion proteins were detected using the commercially available anti-glutathione S-transferase
(GST) antibody (Amrad, Melbourne, Victoria, Australia) at a dilution of
1:5000. The bands were visualized using an horseradish
peroxidase-coupled secondary antibody (Amersham Pharmacia Biotech) or
the ABC system (Vector Laboratories). For Western blotting after the
CaM overlay experiment, blots were stripped using 1% SDS, 1 mM EDTA in Tris-buffered saline.
Nondenaturing Polyacrylamide Gel Shift Assays--
CaM (375 pmol) was incubated with different molar amounts of peptide in a buffer
containing 10 mM HEPES/NaOH (pH 7.2) and 2 mM
CaCl2 or 5 mM EGTA, respectively, for 30 min at
room temperature. The CaM-peptide complexes were then resolved by
nondenaturing gel electrophoresis on 15% gels according to standard
procedures for SDS-polyacrylamide gel electrophoresis, but omitting SDS
and adding 2 mM CaCl2 or 5 mM EGTA,
respectively. Bands were visualized by Coomassie Blue staining.
 |
RESULTS |
Cloning of the Full-length
-Subunit of the Human Rod
Channel--
Previously, we cloned the cDNA for a
-subunit of
the human rod cGMP-gated channel (hRCNC2b; see Ref. 25). Subsequently, this clone appeared to be a truncated form of the cDNA for the full-length
-subunit, with the coded protein missing a segment of
the N terminus (17, 18). Nonetheless, when expressed, the truncated
human protein exhibited all of the hallmark properties of the bovine
full-length protein that was subsequently cloned by Körschen
et al. (18). One of these common properties is the
modulation by Ca2+-CaM. In this study, we obtained the
human full-length channel cDNA to use for experiments. We generated
a probe by performing PCR on cDNA synthesized from bovine retinal
poly(A)+-RNA, using primers based on the 5'-segment (the
GARP region) of the bovine full-length
-subunit (Ref. 24; see also
Ref. 18). This probe was used to screen an adult human retinal cDNA library (see "Experimental Procedures"). Two different types of clones were obtained. One clone, named hGARP1.6 (see Fig.
1), is identical to a human cDNA
clone previously published by Ardell et al. (32) and to the
human t-GARP clone more recently reported by Colville and Molday (33),
except for the absence of six amino acid residues (GAASDP) at position
188 in our hGARP1.6 (indicated by insertion in Fig. 1; see also Refs.
32 and 33). The reason for this difference is unclear, because it
appeared that the same cDNA library was used by all three groups.
The hGARP1.6 clone shares a common 5' region with the other clone,
hGARP2.5, but has a unique 3' region that can also be found in the
genomic DNA for the
-subunit (data not shown), suggesting that it is
a differentially spliced variant. hGARP1.6 was not pursued further. As
for hGARP2.5, its 3'-end shows regions of identity with the 5'-end of
the translated region of hRCNC2b, except that it has an extra insertion
of 264 bases and a unique tail (Fig. 1); furthermore, there is a stop codon 147 bases into the insertion. In the translated protein from this
clone, the C terminus overlaps with the N terminus of hRCNC2b for a
stretch of 117 amino acids, followed by a unique stretch of 49 residues. The overall protein coded by the hGARP2.5 clone has 502 amino
acids and a calculated molecular mass of 55,322 Da. However, in
SDS-gels, the expressed protein showed an apparent molecular mass of
around 110 kDa (Fig. 2A,
hGARP2.5), probably because of its high glutamic acid content.
hGARP2.5 is presumably the human equivalent of the bovine f-GARP
described by Colville and Molday (33).

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Fig. 1.
Structures of different human -subunit
cDNA clones. The relationship among different -subunit
clones is shown, with parallel lines indicating identical
sequences and sloped lines indicating nonhomologous
sequences. The 264-bp insertion in hGARP2.5 is indicated by a
bar. Lines are drawn to scale. hGARP1.6
corresponds to the human t-GARP clone (33) except for the lack of six
amino acids (see "Results" for details). The restriction sites used
for fusion of hRCNC2b and hGARP2.5 are indicated by vertical
bars, and the coding region is illustrated by a shaded
bar. The sequence GAASDP is absent in the translated protein from
our cDNA clones but present in those reported by Ardell et
al. (32) and Colville and Molday (33) (see text).
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Fig. 2.
Heterologous expression of hGARP2.5 and the
full- -subunit. The cDNA for hGARP2.5 or full- was
transfected into HEK 293 cells, and the expressed protein was examined
either biochemically (A and B) or
electrophysiologically (C). A and B,
Western blots of expressed -subunit proteins. Lysates (A
and B) and immunoprecipitates (B) were separated
by SDS-gel electrophoresis, transferred to nitrocellulose, and probed
with the antibody Ab1859. Immunoprecipitations were performed by adding
Ab1859 together with protein A-Sepharose (IP) or as a
covalent conjugate to Sepharose (COV-IP). MOCK,
cells transfected with the expression vector only; full- ,
full- -transfected cells. Arrowheads mark the positions of
the proteins. C, effect of
L-cis-diltiazem on the heteromeric channel
obtained by co-expressing hRCNC and full- . Macroscopic recordings
were made at +60 mV from an excised, inside-out patch of a transfected
cell. 10 µM L-cis-diltiazem
blocked the current by approximately 90%.
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We fused hGARP2.5 and hRCNC2b using Bsu36I and
BamHI restriction sites to produce a single 3.8-kb insert
coding for the complete
-subunit of the human rod channel (see Fig.
1), by comparison to the bovine ortholog (18). The translated protein
has 1245 amino acid residues and a calculated molecular mass of 139,160 Da. The protein, obtained either directly from lysate of transfected HEK 293 cells or by immunoprecipitation from the lysate with an antibody (Ab1859) generated against its GARP part (see "Experimental Procedures"), migrated on an SDS-gel with an apparent molecular mass
of approximately 220 kDa (Fig. 2, A and B). This
full-length clone is identical to that recently identified by Colville
and Molday (33), except again for the missing six residues in our protein described above. Co-transfection of HEK 293 cells with this
full-length
-subunit cDNA and that for hRCNC
(renamed from hRCNC1 of Ref. 26) produced cGMP-activated channels with properties very similar to those observed in co-transfections involving the hRCNC
and hRCNC2b cDNAs. For instance, both types of
heteromeric channels were blocked by
L-cis-diltiazem (see Fig. 2C for
channels containing the full-length
-subunit). Also,
Ca2+-CaM inhibited both types of channels to about the same
extent. This is consistent with the results obtained by Körschen
et al. (18) with the bovine protein. We refer to hRCNC2b as
the trunc-
-subunit and to the full-length
-subunit as the
full-
-subunit.
Inhibition of the
/
-Heteromeric Channel by
Ca2+-CaM--
We first characterized the inhibition of the
/
-heteromeric rod channel by Ca2+-CaM in greater
detail than before (17). The cDNAs for the
- and
trunc-
-subunits were transfected into HEK 293 cells and the macroscopic cGMP-activated current was recorded from excised, inside-out membrane patches. At
60 mV, 60 µM cGMP
evoked a current less than half-maximum, and 250 nM CaM
reversibly inhibited this current by 60-80% in the presence of 50 µM Ca2+ (Fig.
3A). In the absence of
Ca2+, CaM was unable to elicit the inhibitory effect (data
not shown). Also, the recovery of the current from the
Ca2+-CaM inhibition required the removal of both CaM and
Ca2+ (Fig. 3A). Fig. 3B shows the
dose-response relationships, averaged from two patches, between current
activation and cGMP concentration in the absence and presence of
Ca2+-CaM, respectively. In the presence of CaM, the
dose-response relationship was shifted by about 2-fold to higher cGMP
concentrations. This extent of shift is consistent with our previous
finding (17).

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Fig. 3.
Inhibition of the rod / -heteromeric
channel by Ca2+-CaM. In all panels, data were recorded
from excised, inside-out patches of HEK 293 cells transfected with
hRCNC and the trunc- -subunit. A, macroscopic currents
induced by 60 µM cGMP at 60 mV in the presence of 50 µM Ca2+. CaM inhibited the current by
70-80%. Recovery of current required the removal of both CaM and
Ca2+. Results are representative of more than 30 patches.
B, dose-response relationship between activated current and
cGMP concentration in the presence and absence of 250 nM
CaM plus 50 µM Ca2+. Shown are the averaged
data from two patches. Curve fits are according to the Hill equation
(I/Imax = Cn/[Cn + K1/2n]. The K1/2s in
the absence or presence of CaM were 97 and 165 µM cGMP,
respectively, with n = 2.1 for both. C,
current inhibition at different CaM concentrations. Macroscopic
currents were induced by 60 µM cGMP at 60 mV in the
presence of 50 µM Ca2+ and increasing CaM
concentrations. D, dose-dependent inhibition of
the cGMP-activated current by CaM in the presence of 50 µM Ca2+. Shown are the averaged data from 27 cells. One or two low CaM concentrations were tested on each patch, and
the current inhibition was normalized with respect to that obtained
with 250 nM CaM on the same patch. Curve fit is again
according to the Hill equation, with half-maximal inhibition at 4.4 nM CaM and a Hill coefficient (n) of 0.93.
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To investigate the relationship between the extent of channel
inhibition and Ca2+-CaM concentration, we evoked a current
from a patch at
60 mV with 60 µM cGMP and measured the
extent of inhibition of this current at different CaM concentrations in
the presence of 50 µM Ca2+ (Fig.
3C). One or two low CaM concentrations were tested on each patch, and the inhibition of channel activity was normalized with respect to that obtained with 250 nM CaM on the same patch.
Averaged results from 27 experiments indicated that half-maximal
inhibition occurred at a concentration of approximately 4 nM CaM, with a Hill coefficient of 0.93 (Fig.
3D). Because practically all CaM should be
Ca2+-bound in the presence of 50 µM free
Ca2+, Fig. 3D essentially describes the
dependence of channel inhibition on the concentration of
Ca2+-CaM. The value we obtained for half-maximal inhibition
of the channel by Ca2+-CaM is fairly close to that (1-2
nM) previously measured with the native rod channel, either
reconstituted in lipid vesicles or directly from rod outer segment
membranes, using Ca2+ flux as an assay for channel opening
(11, 13).
Channel Protein Binding Studies--
We examined the binding of
CaM to the native
-subunit in human retinal lysate using either
biotinylated-CaM or CaM-coupled alkaline phosphatase as a probe. For
comparison, lysates from bovine retina were also examined in parallel.
For the bovine retinal lysate, the CaM probe recognized a band at a
molecular mass of about 240 kDa in the presence of 1 mM
Ca2+, which is expected for the bovine channel
-subunit
(data not shown). For the human retinal lysate, the CaM probe likewise
recognized a band at a molecular mass of approximately 240 kDa, which
is slightly higher than the 220 kDa expected from the Western blot of
the expressed protein (data not shown; see Fig. 2). It is likely that
this signal resulted from the binding of CaM to a protein other than
the
-subunit. We have not carried out CaM binding experiments with
the immunopurified channel protein from human retinal lysate because of
the scarcity of tissue. However, we performed similar experiments with
the Ab1859-immunoprecipitated full-
-subunit expressed in HEK 293 cells (see Fig. 2B) and failed to detect a binding signal.
This could imply that the CaM affinity of the expressed
-subunit is
very weak and therefore not detectable, or that under the gel overlay
conditions, the human protein had not renatured enough to permit
significant CaM binding. The intense band in Fig. 2B between
106 and 205 kDa is probably due to proteolytic cleavage of the
full-length
-subunit.
We have repeated the same experiments using GST fusion proteins of the
N and C termini of the
-subunit. For the N-terminal fusion protein,
the N terminus of the trunc-
-subunit was used instead of the
full-
-subunit, simply because it was shorter. Because the
trunc-
-subunit confers the same CaM effect as the full-
-subunit,
this point is immaterial. Both the N- and C-terminal fusion proteins
were found to bind CaM when probed with biotinylated-CaM or CaM-coupled
alkaline phosphatase in gel overlay assays (Fig. 4; see also Figs.
5D and 8B).
However, their affinities for CaM were much weaker than that observed
for the GST-fusion protein of the N terminus of the
-subunit of the
olfactory cyclic nucleotide-gated channel studied in parallel (Fig. 4,
N-OCNC
), which previous work has shown to have a high
affinity for CaM (22). On the other hand, the much weaker CaM affinity
of the C-terminal fusion protein compared with the N-terminal fusion
protein (Fig. 4) could have resulted from a lower blotting efficiency,
as suggested by Western blots (data not shown). We found that the N-
and C-terminal fusion proteins of the trunc-
-subunit retained the
binding to CaM even in the absence of Ca2+ (Fig. 4,
right panel). This finding could have suggested the presence
of Ca2+-independent binding sites for CaM on the N and C
termini. However, even for the GST-fusion protein of the N terminus of
the olfactory channel
-subunit, some CaM binding was retained in the
absence of Ca2+. Furthermore, the experiments described
below with peptides corresponding to the putative binding sites on the
two termini indicated that these peptides required Ca2+ to
bind CaM. These observations, together with the Ca2+
requirement for the functional modulation of the
/
-heteromeric channel complex described earlier, indicate that the apparent Ca2+-independent CaM binding of the fusion proteins was
possibly an artifact of the experimental conditions. Contradictory
observations on the Ca2+ dependence of CaM binding under
different conditions has been reported for the Ras-like GTPase RIC,
which required Ca2+ to bind CaM in a gel overlay but did
not require Ca2+ to bind to CaM-agarose beads in solution
(34). We have not pursued this point further. In any case, no strong
binding of CaM to the
-subunit could be found for the heterologously
expressed protein, either in its entirety or as fusion proteins,
although this may be due to incomplete renaturation.

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Fig. 4.
CaM binding of fusion proteins of the
trunc- -subunit. GST-fusion proteins of the trunc- -subunit
were resolved by SDS-polyacrylamide gel electrophoresis and transferred
to nitrocellulose. To prevent bleeding, the blot was cut into four
pieces and then probed with biotinylated CaM in the presence or absence
of Ca2+. After the CaM overlay, blots were stripped and
probed with the -GST antibody, and the results indicated that the
N-terminal fusion protein was transferred to the blot more efficiently
than the C-terminal fusion protein (data not shown).
N-OCNC , N-terminal fusion protein of the -subunit of
the rat olfactory cyclic nucleotide-gated channel; N-2b,
N-terminal fusion protein of the trunc- -subunit; C-2b,
C-terminal fusion protein of the trunc- -subunit. Arrows
indicate the positions of the fusion protein bands: 1, N-2b;
2, C-2b; 3, N-OCNC .
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Fig. 5.
CaM-binding of C-terminal peptides and fusion
proteins. A, schematic diagram of the trunc- -subunit
showing the locations of the various C-terminal peptides. Gray
boxes represent the transmembrane domains, and the striped
region represents the pore region (P). The
arrowhead indicates a region containing putative CaM-binding
sites upstream of the cyclic nucleotide-binding site (CN)
that was not examined (see text). The sequences of the peptides are
shown with numbers indicating the first and last residues.
B, gel shift experiments with the C-terminal peptides. CaM
(375 pmol) was incubated with the peptides KY13, KY14, KY15,
and KY19 in different mole ratios (peptide:CaM) in the
presence of 2 mM Ca2+, and the peptide-CaM
complexes were resolved on a 15% nondenaturing gel and visualized by
Coomassie Blue staining. The left lane contains CaM only.
The arrowhead indicates the position of unbound CaM.
C, fluorescence measurements of the interaction between the
C-terminal peptides and CaM. Peptides were incubated with dansyl-CaM at
increasing mole ratios, and the fluorescence at 480 nm was measured in
the presence of 0.5 mM Ca2+; excitation
wavelength was at 340 nm and bandwidths were 10-15 nm. The normalized
fluorescence increase at 480 nm (representing the fraction of bound
peptide) was plotted against the concentration of free peptide (see
text). Open triangles represent data from two experiments in
the presence of 100 nM dansyl-CaM, and filled
circles represent data from two or three experiments in the
presence of 300 nM dansyl-CaM. Curves are fitted according
to the Hill equation, with Kd and n
values of 2.9 µM and 1.1 (KY13), 0.4 µM and
1.2 (KY14), 0.3 µM and 1.1 (KY15), and 14.5 µM and 0.8 (KY19), respectively. For KY14 and KY15, the
data with 100 nM CaM were used for curve fits. The
experiments with 100 nM dansyl-CaM had the drawback of
giving weaker fluorescence signals, but they gave more reliable
Kd measurements for peptides with higher CaM
affinity. D, gel overlay of C-terminal fusion proteins with
biotinylated CaM. C-2b, wild-type C-terminal fusion protein
of the trunc- -subunit; C-2bMG8, same fusion protein with
deletion of MG8, corresponding to the region covered by KY14, KY15, and
KY19. Probing of the same (stripped) blot with an -GST-antibody gave
bands of about equal intensity for C-2b and C-2bMG8 (positions
indicated by arrowheads).
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The C Terminus of the
-Subunit Does Not Contain a CaM-binding
Site Crucial for the CaM-mediated Inhibition--
We scanned the N-
and C-terminal sequences of the trunc-
-subunit and identified
potential binding sites for CaM in both locations, based on the
presence of conserved hydrophobic residues within short stretches of
amino acid sequence (35, 36). The sites on the C terminus are more
prominent, so we focused on them first. On the C terminus, there are
two potential regions, one upstream and the other downstream of the
cyclic nucleotide-binding domain. The first region (see Fig. 5,
arrowhead), corresponding to residues Ile629 to
Leu646 and containing two consensus motifs, was ignored
because this region bears strong homology to the corresponding region
in the
-subunit of the rod channel (26), a protein known not to bind or be inhibited by CaM (10, 11, 17, 22). For the second region, we
synthesized a peptide, KY19 (Ala781-Lys804;
see Fig. 5A), and performed gel shift experiments. The
peptide and CaM were loaded in different mole ratios onto a
nondenaturing gel to examine the ability of the peptide to bind CaM and
retard its migration. In the absence of Ca2+, KY19 did not
retard the migration of CaM (data not shown). In the presence of 2 mM Ca2+, a mixture of KY19 and CaM at a ratio
of 50:1 showed a limited ability to retard the migration of CaM (Fig.
5B). To measure the affinity between KY19 and CaM, we
employed the fluorescent CaM derivative, dansyl-CaM. When a peptide is
bound to dansyl-CaM, the fluorescence increases and shifts to shorter
wavelengths (22, 37). The relationship between the fraction of bound
dansyl-CaM and the concentration of free peptide measured in these
fluorescence experiments can be approximately described by a binding
isotherm corresponding to the Hill equation with a coefficient of unity and a dissociation constant, Kd, of approximately 15 µM (Fig. 5C). To be certain that this low
affinity did not result from an incomplete CaM-binding site on the
peptide, we examined three other peptides, KY13, KY14, and KY15,
spanning adjacent regions also rich in hydrophobic and positively
charged amino acid residues (see Fig. 5A). KY13 and KY15 had
a somewhat higher ability to retard CaM migration, but the effect was
still weak (Fig. 5B). In fluorescence experiments with
dansyl-CaM, the measured Kd was approximately 3 µM for KY13, 0.4 µM for KY14, and 0.3 µM for KY15 (Fig. 5C). As in the gel shift
experiments, these peptides did not appear to interact with dansyl-CaM
in the absence of Ca2+.
We examined deletion mutants of the C-terminal fusion protein for their
ability to bind CaM in gel overlay experiments. When a mutant lacking
the entire region downstream of the cyclic nucleotide-binding site was
expressed in BL21 cells, the protein was hardly expressed. Another
mutant (MG8) lacking the region (residues 749-805) that includes the
combined KY14, KY15, and KY19 lost the ability to bind CaM (Fig.
5D). Thus, it appears that there is a specific Ca2+-CaM-binding site on the C terminus covered by these
peptides, but the affinity of this site for CaM is weak.
We tested the functional importance of this CaM-binding site by
generating deletion mutants of the trunc-
-subunit lacking the region
corresponding to KY13, KY19, or the combined region of KY14, KY15, and
KY19. When co-expressed with hRCNC
, the mutant lacking KY13 did not
seem to express well, as suggested by the lack of blockage of the
cGMP-activated current by L-cis-diltiazem (data
not shown). However, when the mutants lacking KY19 (MG2) or KY14, KY15,
and KY19 together (MG8) were co-expressed with hRCNC
, the resulting
cGMP-activated current was still inhibited by CaM to a similar extent
as the wild-type (Fig. 6). Thus, the CaM-binding site identified in the region spanning KY14, KY15, and KY19
on the C terminus does not appear to be necessary for the modulation of
the heteromeric channel by CaM, but the possibility cannot be excluded
that it contributes to the high sensitivity of the rod channel
heteromeric channel to CaM, as observed in the electrophysiological
experiments.

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Fig. 6.
Retention of the CaM-inhibition of the
/ -heteromeric channel containing the C-terminal deletion mutants
MG2 (A) and MG8 (B) of the
trunc- -subunit. Macroscopic currents evoked by 60 µM cGMP at 60 mV from excised, inside-out patches of
HEK 293 cells transfected with hRCNC and the mutants MG2
(A) and MG8 (B). Results are representative of 3 (MG2) and 12 (MG8) patches. In both panels, the
numbers in the schematic diagram of the trunc- -subunit
indicate the first and last residues of the domain deleted (indicated
by a black box).
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The N Terminus of the
-Subunit Contains a Region Crucial for the
CaM-mediated Inhibition--
Because the experiments on the C terminus
indicated that not every CaM-binding site is necessarily involved in
the modulation of the rod channel, we adopted a functional assay to
study the N terminus. A number of deletion mutants were generated
spanning consecutive regions of the N terminus of the trunc-
-subunit
(Fig. 7A) and were
co-expressed individually with hRCNC
. To test for a loss of
CaM-modulation of the heteromeric channel expressed in HEK 293 cells,
we performed electrophysiological recordings. Among these, we found
that only mutants MG4 and BA104, which lacked a region close to the
first transmembrane domain, had lost the inhibitory effect (Fig.
7B). This region is also rich in hydrophobic and basic
residues (Fig. 8A).
Experiments on the full-
-subunit lacking the region corresponding to
MG4 gave the same result. Sequence scanning suggests that the region
deleted in MG17 (see Fig. 7A) may also contain a CaM-binding
site, but this deletion mutant retained the CaM-mediated inhibition
(Fig. 7B).

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Fig. 7.
Effects of N-terminal deletions in the
trunc- -subunit on the Ca2+-CaM inhibition of the
/ -heteromeric rod channel. A, locations of various
N-terminal deletions of the trunc- -subunit are indicated by
black boxes. The numbers indicate the first and
last residues of each deleted domain. B, macroscopic
currents induced by 60 µM cGMP at 60 mV from excised,
inside-out patches of HEK 293 cells transfected with hRCNC and
BA101, MG3, MG9, MG4, BA104, and MG17. Records are representative of
3-10 experiments for each deletion mutant.
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Fig. 8.
CaM-binding of N-terminal peptides and fusion
proteins. A, schematic diagram of the trunc- -subunit
showing the locations of the two N-terminal peptides. The
arrowhead indicates the end of the GARP-region.
Below the diagram, the sequences of the peptides MEG1 and
MEG2 are shown with numbers indicating the first and last
residues. B, CaM overlay of N-terminal fusion proteins
probed with biotinylated CaM. N-2b, wild-type N-terminal
fusion protein of the trunc- -subunit; N-2bMG4, the same
fusion protein with the deletion MG4 (see Fig. 7). Probing of the same
(stripped) blot with an -GST-antibody gave bands of about equal
intensity for N-2b and N-2bMG4 (positions indicated by
arrowheads). C, gel shift experiments with the
N-terminal peptides. CaM (375 pmol) was incubated with the peptides
KY9, RH106, MEG1, and MEG2 in different mole ratios in the presence of
2 mM Ca2+ (see text for a description of KY9
and RH106). The peptide-CaM complexes were resolved on a 15%
nondenaturing gel and visualized by Coomassie Blue staining. The
left lane contains CaM only. The arrowhead
indicates the position of unbound CaM. D, fluorescence
measurements of the interaction between the N-terminal peptides and
CaM. Peptides were incubated with dansyl-CaM at increasing mole ratios
in the presence of 0.5 mM Ca2+, and the
fluorescence at 480 nm was measured; excitation was at 340 nm, and
bandwidths were 10-15 nm. The normalized fluorescence increase
(representing the fraction of bound peptide) was plotted against the
concentration of free peptide (see text). Open triangles
represent data from two experiments in the presence of 100 nM dansyl-CaM, and filled circles represent data
from three or four experiments in the presence of 300 nM
dansyl-CaM. Curves are fitted according to the Hill equation, with
Kd and n values of 3.6 µM
and 1.2 (MEG1), 14.4 µM and 1.3 (MEG2), and 29 nM and 1.0 (RH106), respectively. The experiments with 100 nM dansyl-CaM had the drawback of giving weaker
fluorescence signals, but they gave more reliable Kd
measurements for the RH106 peptide, which had a higher CaM affinity. No
difference between using 100 or 300 nM dansyl-CaM was
observed for MEG1 and MEG2.
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A fusion protein of the trunc-
-subunit N terminus with a deletion
corresponding to MG4 likewise lost its binding to CaM (Fig. 8B). Two peptides, MEG1 and MEG2, covering the deleted
region were synthesized and tested for their ability to bind CaM. In a
gel shift assay, both peptides appeared to bind CaM, but again very
weakly (Fig. 8C). As controls, we examined several other peptides in the same experiment. One peptide, KY9, corresponds to the
CaM-binding site on the N terminus of the
-subunit of the olfactory
cyclic nucleotide-gated channel and binds Ca2+-CaM with
nanomolar affinity (22). It retarded the migration of CaM completely at
a peptide:CaM ratio of 2:1. Another peptide, RH106, corresponds to one
of the CaM-binding sites on a
N-methyl-D-aspartate receptor channel subunit
(NR1) and has previously been shown to bind CaM with a
Kd of lower than 100 nM (38). RH106, likewise, shifted CaM according to the published results. In
experiments with dansyl-CaM, the measured Kd for
both MEG1 and MEG2 was in the range of 3-10 µM, compared
with a Kd of approximately 30 nM for
RH106, which is consistent with the published value. A third control
peptide, covering a region in the N terminus of the olfactory channel
-subunit and not able to bind CaM (KY8; see Ref. 22), neither
shifted the CaM band in a gel shift assay nor showed any binding to
dansyl-CaM at up to micromolar concentrations (data not shown). Thus,
the CaM binding ability of MEG1 and MEG2 seems genuine, albeit weak.
This CaM binding was Ca2+-dependent.
These results indicate that a region on the N terminus of the
-subunit is crucial for the CaM-mediated inhibition of the human rod
channel. When this region is removed, the ability of the N-terminal
fusion protein to bind CaM disappears, as does the CaM-modulation of
the heteromeric channel. However, as determined with fusion proteins
and peptides, the Ca2+-CaM-affinity for this binding site
is surprisingly low, being almost 1000-fold lower than the apparent
affinity measured in the electrophysiological experiments.
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DISCUSSION |
Based on Ca2+-flux measurements, others have shown
that in the presence of Ca2+, CaM inhibits the native rod
cGMP-gated channel by shifting the dependence of its activation on cGMP
to higher cGMP concentrations; the half-maximal inhibition of the
current at low cGMP concentrations occurs at 1-2 nM CaM
(10, 11, 13). Our electrophysiological recordings described here, on
excised, inside-out patches of HEK 293 cells transfected with the
cloned human cGMP-activated channel
- and
-subunits, have led to
a similar conclusion, giving a half-maximal inhibition at approximately
4 nM CaM. These values suggest a high apparent affinity of
Ca2+-CaM for the rod channel. By contrast, our CaM overlay
experiments on human retinal lysates and the heterologously expressed
human
-subunit, which is known to confer the CaM effect (Ref. 17; see also Ref. 18), have failed to detect any obvious binding to CaM.
The lack of CaM binding to human retinal lysates could have been due to
the limited amount of tissue in the experiments or to the degradation
of the tissue, which was obtained several hours post-mortem. However,
these explanations cannot account for the lack of CaM binding to the
heterologously expressed
-subunit, which we were able to harvest
from transfected cells in higher quantity and with presumably little
degradation. Previously, iodinated (125I) CaM was used as a
probe in gel overlay experiments for detecting CaM binding to the
native bovine rod channel
-subunit (10, 11) and to the
heterologously expressed rat olfactory channel
-subunit (22),
whereas biotinylated CaM and CaM-coupled alkaline phosphatase were used
in our experiments. Because the latter two reagents have been shown to
be as sensitive as, or more sensitive than, iodinated CaM in overlay
assays on hair bundles of cochlear hair cells (31), it seems unlikely
that our negative binding results have resulted from insensitive
probes. More probably, insufficient renaturation of the human
-subunit under the gel overlay conditions led to undetectable CaM
binding.
In experiments with the GST-fusion proteins of the trunc-
-subunit N
and C termini, we did detect CaM binding, but the signals were very
weak when compared with that for the N-terminal fusion protein of the
olfactory cyclic nucleotide-gated channel
-subunit, which has a high
affinity site for CaM (22). These weak affinities with fusion proteins
were consistent with the results from gel shift and fluorescence
experiments with synthetic peptides corresponding to the binding sites
on the two termini, with the fluorescence experiments giving
dissociation constants in the micromolar range. The low affinity of the
identified C-terminal CaM-binding site may be genuine, especially
considering that deletion mutants of the
-subunit lacking this
region still conferred CaM-modulation to the
/
-heteromeric
channel complex. The low affinity of the N-terminal CaM-binding site is
more surprising, however, because from deletion studies this is the
site that is crucial for the CaM-modulation of the channel. Its
affinity for CaM (Kd of 3-10 µM) as
measured with synthetic peptides is approximately 1000-fold lower than
the half-maximal inhibition constant of 4 nM CaM derived
from the electrophysiological experiments. From previous work on the
olfactory channel, it is clear that CaM affects the gating of the
channel (22). Assuming that CaM acts on the rod channel with a similar
mechanism (see below), the apparent affinity of CaM for the intact
channel protein can, in principle, be enhanced by the coupling between
the ligand-binding and channel-gating steps. However, because of the
relatively weak modulation of the rod channel by CaM (approximately
3-fold current reduction at low cyclic nucleotide concentrations; see
"Results") compared with that for the olfactory channel (over
100-fold current reduction by CaM; see Refs. 21 and 22), it seems
unlikely that this binding-gating coupling can lead to a 1000-fold
increase in the apparent affinity for CaM. Moreover, despite the strong
modulation of the olfactory channel by CaM, the CaM affinity for the
binding site on this channel as measured with peptide-binding assays is not any lower than that measured with the electrophysiological experiments on the current inhibition (see below). There are several possible explanations. One is that the N-terminal peptides were structurally unable to reproduce the binding properties of the whole
protein. Even though the peptides we synthesized are over 20 amino acid
residues in length, they may still be not long enough to reproduce the
native binding site. For the adenylyl cyclase in the bacterium
Bordetella pertussis, for example, it was found that an
increase in the length of a peptide spanning the CaM-binding site
beyond 43 residues continues to increase its affinity for CaM (39, 40).
Another possibility is that more than one
-subunit is present in the
/
-heteromeric channel complex and that CaM binds to them with
positive cooperativity. Based on Ca2+-flux measurements,
Bauer (13) suggested that two CaM molecules bind to a native rod
channel complex. In our experiments on the heterologously expressed
channel, on the other hand, the relationship between current inhibition
and CaM concentration had a Hill coefficient near 1 (see Fig. 3), which
suggests that perhaps only one CaM molecule binds to the channel
complex. Because the two studies employed different preparations and
different measurements, a strict comparison between them may not be
meaningful. Nonetheless, considering that there is no known precedence
for cooperative binding of CaM to a target protein, such a scenario is
probably unlikely in our situation. A third, and more realistic,
possibility is that even though the identified domain on the N terminus
is a bona fide CaM-binding site, other regions on the
channel protein facilitate or stabilize this binding through
allosteric, electrostatic, or hydrophobic interactions. In the skeletal
muscle phosphorylase kinase, for example, it has been suggested that
two physically separate domains interact simultaneously with a single
CaM molecule (41, 42). This situation can be considered to be a more
extreme variation of the first possibility mentioned above. In
principle, other domains on the rod channel
-subunit participating
in the interaction with CaM can be somewhere on the N terminus, as far away as in the C terminus, or even located in other subunits of the
oligomeric channel. In this respect, the rod channel differs from the
olfactory channel. Half-maximal inhibition of the current through the
olfactory channel at low ligand concentrations occurs nominally at 4-5
nM (this being calculated as the square root of the value
of 21 nM CaM in Fig. 3 of Ref. 21, based on a Hill coefficient of 2 for the activation of the channel by cGMP), which matches quite well the Kd of 3-4 nM for
the peptide corresponding to the CaM-binding site on the
-subunit of
the channel (22). Thus, for the olfactory channel, other regions
besides the binding site may be minimally involved in interacting with
CaM.
Despite the apparent difference in CaM-binding characteristics between
the rod and olfactory channels, it is interesting to note that the
functionally important CaM-binding site is situated at about the same
location on both channels, both being a short distance N-terminal of
the beginning of the first putative transmembrane domain (Fig.
9). Nonetheless, the binding site for the
rod channel is situated on the
-subunit, but on the
-subunit for
the olfactory channel. In comparison, the
-subunit of the rod
channel neither binds CaM (Ref. 10; see also Ref. 22) nor is modulated
by CaM (17). As for the
-subunit of the olfactory channel, it also
does not confer any CaM modulation to the heteromeric channel complex
when co-expressed, for example, with the rod channel
-subunit (43),
although whether it binds CaM has not yet been studied. Finally, it
should be mentioned that the
-subunit of the cone channel also has a
high affinity CaM-binding site in a homologous position on its N
terminus (44);2 surprisingly,
however, there is no CaM modulation at least of the homomeric channel
formed by this subunit (Ref. 44; see also Ref. 45).2 Taken
together, these observations suggest an evolutionary path in which the
ancestral channel that gave rise to the various
- and
-subunits
of the cyclic nucleotide-gated channels might already have a
CaM-binding site-like domain on its N terminus. As the various subunit
species arose over time, the CaM-binding capacity of the domain evolved
as well, becoming either stronger or weaker in the different subunits.
In parallel, other regions of the subunits evolved independently, such
that a functional modulation by CaM occurs only when a CaM-binding site
and other interacting regions on the channel are simultaneously present
(as is the case with the olfactory channel
-subunit; see below). A
sequence alignment of the CaM-binding sites on the olfactory channel
-subunit and the rod channel
-subunit is shown in Fig. 9. The
CaM-binding site on the olfactory channel
-subunit conforms well to
both the 1-8-14 and 1-5-10 motifs characteristic of many CaM-binding sites, where the numbers indicate the positions of key aromatic or
long-chain aliphatic residues separated by other residues, including
some positively charged ones (36). At the same time, the binding site
shows a basic amphiphilic structure in a Kyte-Doolittle plot, with a
net charge of +2 (46, 47). Both the hydrophobic and positively charged
residues are thought to interact with CaM (48). The CaM-binding site on
the rod channel
-subunit, on the other hand, shows only partial
resemblance to these motifs. For example, although the residues in
positions 1, 5 and 8 are aromatic or hydrophobic in nature, residue 14 is not (although residue 15 is). At the same time, the sequence has a
net charge of only +1. Finally, a Kyte-Doolittle plot of the sequence
does not show obvious amphiphilicity. These latter features may account for the relatively weak CaM affinity found for the peptides and fusion
proteins.

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Fig. 9.
Sequence alignment of the CaM-binding sites
in the olfactory channel -subunit and the rod channel
-subunit. Schematic representation of rOCNC and the
trunc- -subunit showing the transmembrane regions
(shaded), the pore region (P)
(hatched), and the CaM-binding site (black).
Sequences of the CaM-binding sites are aligned, and conserved residues
are indicated by vertical bars.
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The detailed mechanism by which CaM modulates the rod channel remains
unclear. For the olfactory channel, CaM binds to a domain on the N
terminus of the
-subunit that, in the absence of CaM, promotes the
open state of the channel (Ref. 22; see also Refs. 15 and 49). When
this domain is deleted, or when CaM binds to it, the influence of this
domain in promoting the open state of the channel is lost, consequently
leading to an inhibition of the current (22). Most recently,
co-immunoprecipitation experiments have indicated that the N and C
termini indeed directly interact with each other, and this interaction
is disrupted by the binding of CaM to the N terminus of the channel
(Ref. 23; see also Ref. 50). Thus, it appears that this
N-terminal-C-terminal interaction promotes the opening of the channel.
As for the rod channel, because the modulation by CaM is relatively
weak (see above), we have not, unfortunately, been able to conclusively
demonstrate whether or not a deletion mutant of its
-subunit lacking
the CaM-binding site (MG4) behaves like the wild-type with CaM bound,
that is, with a shift of the cGMP dose-response relationship to higher cGMP concentrations (data not shown). By analogy to the olfactory channel, however, the mechanism may still be similar.
We thank P. L. Pedersen for use of the
spectrophotometer and T. R. Golden for assistance and advice. We
are grateful to A. R. Rhoads for sharing the motif scanning
program Seqchrx, J. Nathans for the human retinal cDNA library, and
T. W. Kraft for human retinal tissue. We also thank R. S. Molday for providing us with the cDNA clone for the bovine
full-length
-subunit, which we used for Western blots, and for
helpful suggestions. We thank R. D. Barber, Y. Koutalos, and D. Krautwurst for valuable comments on the manuscript and A. G. Betz
for help with artwork. We thank J. Lai, J. Li, and M. Dehoff for
technical support. Finally, we thank U. B. Kaupp and colleagues
for discussions and for communicating data prior to publication.
While this work was in progress, U. B. Kaupp
brought to our attention that, in his laboratory, broadly similar
results had been obtained for the
-subunit of the bovine rod cyclic
nucleotide-gated channel (U. B. Kaupp, personal communication).