Department of Molecular and Cellular Biology, Harvard
University, Cambridge, Massachusetts 02138
Cyclic
nucleotide-gated cation channels in skeletal muscle are responsible for
insulin-activated sodium entry into this tissue (J. E. M. McGeoch and
G. Guidotti. J. Biol. Chem. 267:
832-841, 1992). These channels have previously been isolated from
rabbit skeletal muscle by 8-bromoguanosine 3',5'-cyclic
monophosphate (8-BrcGMP) affinity chromatography, which separates them
into two populations differing in nucleotide affinity [L. C. Santy and G. Guidotti. Am. J. Physiol.
271 (Endocrinol. Metab. 34): E1051-E1060, 1996]. In this study, a polymerase chain reaction approach was used to identify skeletal muscle cyclic nucleotide-gated channel cDNAs. Rabbit skeletal muscle expresses the same cyclic nucleotide-gated channel as rabbit aorta (M. Biel, W. Altenhofen, R. Hullin, J. Ludwig, M. Freichel, V. Flockerzi, N. Dascal, U. B. Kaupp,
and F. Hofmann. FEBS Lett. 329:
134-138, 1993). The entire cDNA for this gene was cloned from
rabbit skeletal muscle and an antiserum to this protein produced.
Expression of this cDNA produces a 63-kDa protein with cyclic
nucleotide-gated channel activity. A similarly sized immunoreactive
protein is present in sarcolemma. Purification of the expressed
channels reveals that this single gene produces both native skeletal
muscle channel populations.
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INTRODUCTION |
CYCLIC NUCLEOTIDE-GATED (CNG) channels were first
identified in rod outer segments, where they are responsible for the
dark current (9). It was subsequently shown that similar channels are
also present in olfactory receptor cilia (25). In both of these
tissues, the CNG channels are involved in generating an electrical
signal in response to sensory stimuli (18). Purification of the CNG
channel from rod outer segments was followed by the cloning of the CNG
channel gene (4, 19). Homologous genes comprising a large family have
been cloned from olfactory receptors, retinal cones, and a variety of
nonsensory tissues (6, 7, 20). The CNG channel family members contain a
putative cyclic nucleotide binding domain in their COOH terminal end
and have homology to the voltage-gated cation channel family (16, 19, 30).
Although the CNG channels in photoreceptor and olfactory receptor cells
have a clearly defined physiological role, the role of CNG channels
expressed in most nonsensory tissues is less clear. Because CNG
channels conduct Ca2+, one
possible function of these channels may be to increase internal calcium
levels in response to second messengers (10, 18). For example, a CNG
channel may mediate sperm chemotaxis, which depends upon activation of
a membrane- bound guanylate cyclase and entry of extracellular
Ca2+ (12). The gene encoding the
cone CNG channel is also expressed in the testis, and sperm membranes
have been shown to contain this protein, suggesting that this channel
may connect guanosine 3',5'-cyclic monophosphate (cGMP) to
Ca2+ entry (10, 29). Many other
nonsensory tissues have been shown to express CNG channel genes.
However, investigation of physiological roles or expression of the
proteins in most of these tissues has not been undertaken (10).
A physiological role for CNG channels has been studied in skeletal
muscle. In this tissue, CNG channels seem to be responsible for
insulin-activated sodium entry (22, 23). Release of insulin into the
blood promotes uptake of potassium into skeletal muscle and adipocytes
by increasing the activity of the
2 isoform of the Na-K-ATPase
(3, 21, 27). Uptake of potassium from the blood into skeletal muscle
reflects the ability of this tissue to act as a potassium reservoir for
the rest of the body (2). To prevent depletion of internal sodium by
the activated Na-K-ATPase, insulin also increases sodium entry into
skeletal muscle (3, 15). This constant replenishment of internal sodium
allows the increased Na-K-ATPase activity to be maintained for upwards
of an hour (3, 23, 27).
An insulin-sensitive cation channel has been identified in rat skeletal
muscle by patch clamping (22). This channel is activated by the
combination of insulin and GTP or by cGMP alone, suggesting that it is
a member of the CNG channel family. Similar to other members of the CNG
channel family, this channel is fairly nonselective for monovalent and
divalent cations and displays a flickering block by calcium (22, 30).
An inhibitor of the skeletal muscle CNG channel, µ-conotoxin GIIIB,
can block insulin-activated sodium entry into intact skeletal muscle
(22, 23). Two forms of CNG channel activity have been isolated from
rabbit skeletal muscle using 8-bromoguanosine 3',5'-cyclic
monophosphate (8-BrcGMP) affinity chromatography and reconstituted into
liposomes (28). These CNG channel forms differ in their activation by
cyclic nucleotides. One form has a half-maximal activation constant
(K1/2)
for cGMP of 5.79 × 10
7 M and a Hill
coefficient of 3.63, whereas the other form has a lower affinity with a
K1/2 of 1.93 × 10
5 M and a Hill
coefficient of 1.16 (28). The two forms of CNG channel may be the
products of different CNG channel genes, the consequence of
differential splicing of a single gene, or the result of modulation of
a single protein. In understanding insulin-activated sodium entry via a
CNG channel, it will be important to know which of these possibilities
is responsible for the two forms present in skeletal muscle.
In this study, polymerase chain reaction (PCR) has been used to
identify and clone the rabbit skeletal muscle CNG channel cDNA. This
cDNA is identical to one previously cloned from rabbit aorta (1). An
antiserum, anti-rabbit-cyclic-nucleotide-gated channel 3-1
(
rCNG3-1), was raised against the COOH-terminal
145 amino acids of the rabbit skeletal muscle CNG protein. Expression of this gene in tissue culture cells leads to the production of a
63-kDa protein that reacts with the
rCNG3-1 antiserum. A
protein of the same size present in rabbit skeletal muscle sarcolemma also reacts with this antiserum. Expression of the skeletal muscle CNG
channel cDNA in tissue culture cells produces CNG channel activity that
is most similar to the low cGMP affinity form of the native skeletal
muscle channel. This CNG channel activity is inhibited by µ-conotoxin
GIIIB, which has previously been shown to inhibit the native skeletal
muscle CNG channel and to inhibit insulin-activated sodium entry into
this tissue (22). Isolation of the expressed CNG channels with 8-BrcGMP
affinity chromatography reveals two forms of CNG channels that resemble
the native skeletal muscle channels. This result suggests that the
single CNG channel gene cloned from skeletal muscle is responsible for
both forms of channel observed in this tissue and that the two forms
may arise by differential modulation of a single CNG channel protein.
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MATERIALS AND METHODS |
Materials. Restriction enzymes and
other enzymes for molecular biology were from New England Biolabs
(Beverly, MA). Glutathione-Sepharose, thrombin,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), µ-conotoxin GIIIB, and protease inhibitors were purchased from Sigma (St. Louis, MO). All reagents for tissue culture were purchased from GIBCO-BRL (Grand Island, NY). HEK 293T cells, which stably express SV40 large T, were obtained from the laboratory of Dr.
Ernest Peralta (Harvard University, Cambridge, MA). Lipids were
purchased from Avanti Polar Lipids (Alabaster, AL). Primers were
ordered from Genemed Biotechnologies (South San Francisco, CA).
RNA isolation and cDNA production.
Total RNA was isolated from rabbit tissues using acid guanidinium
thiocyanate phenol extraction (26). mRNA was isolated from the total
RNA using the PolyATtract mRNA isolation system (Promega, Madison, WI)
or the polyA Spin mRNA isolation kit (New England Biolabs) according to
the manufacturer's instructions. The mRNA was transcribed into first
strand cDNA using oligo(dT), random 9-mer primers, and Stratascript
Reverse Transcriptase (Stratagene, La Jolla, CA) according to the
manufacturer's protocol.
PCR. Primers were designed to
correspond to conserved regions of the cyclic nucleotide binding domain
of CNG channels or to the published sequence of the rabbit aorta CNG
channel gene (1). All primers, except 955, contained restriction enzyme
sites at their 5'-end (DCNGF1 and DCNGF2:
EcoR I; all other primers:
BamH I). Primer sequences are given
below. Degenerate positions are given in parentheses and I stands for
inosine.
PCR
reactions used cDNA as template for amplification with the above
primers. Generally, 35 cycles of PCR were run for each reaction.
Reactions contained 2.5 units of AmpliTaq (Perkin-Elmer, Foster City,
CA) and 0.0025 units Pfu (Stratagene) polymerases. Reactions to identify the gene (Fig.
1A)
were annealed at 50°C and had 2 min of extension at 72°C.
Reactions to isolate the gene (Fig.
1C) were annealed at 57°C and
had 5 min extension at 72°C. Aliquots of this primary amplification
were used as template and reamplified using the same primers and
conditions to obtain enough DNA for cloning. PCR reactions to determine
tissue expression (Fig. 2) had an annealing
temperature of 55°C and 2 min extension at 72°C. Reaction
products were purified with the QIAquick PCR purification kit (Qiagen,
Chatsworth, CA). Reaction products were then digested with the
restriction enzymes whose sites were present at the 5'-end of the
primers, separated by agarose gel electrophoresis, isolated with the
Geneclean or MERmaid DNA isolation kits (Bio101), and ligated into
pGEM3zf
(Promega).

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Fig. 1.
Polymerase chain reaction (PCR) identification and cloning of the
rabbit skeletal muscle cyclic nucleotide-gated (CNG) channel gene.
A: PCR amplification of skeletal
muscle CNG gene, using mRNA (0.35 µg) or cDNA (made from 0.25 µg
mRNA) as template with degenerate primers DCNGF1, DCNGF2, and DCNGR.
These primers correspond to the amino acid sequences (I/V)G(R/K)EMYI
and GNRRTAN, which are conserved in the cyclic nucleotide binding
domains of all known CNG channels. B:
sequence alignment of the skeletal muscle CNG gene. Skeletal muscle PCR
product was cloned and sequenced. Analysis of the sequence revealed
that it was identical to the previously cloned rabbit aorta CNG channel
gene (1). C: cloning of the entire
skeletal muscle CNG channel gene by PCR from rabbit skeletal muscle
cDNA with primers based on the published sequence of the rabbit aorta
CNG channel. Lane 1: 3'-end of
the CNG channel gene amplified with primers DCNG51 and RACNG32 (aorta
sequence bases 1120 to 2669). Lane 2:
5'-end of the gene amplified with primers RACNG599 and 5128 (bases 99 to 1933).
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Fig. 2.
Tissue expression of skeletal muscle CNG channel gene. Rabbit mRNA (5 µg) from the indicated tissues was transcribed into cDNA, and
one-fifth of the cDNA was used as template for PCR with primers 955 and
5128 (bases 955 to 1933, sequences described in MATERIALS AND
METHODS). Positive control contains the cloned skeletal muscle
CNG channel gene as template, whereas negative control contains no
template.
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DNA sequencing analysis and alignment.
PCR products were sequenced after cloning into pGEM. Sequencing was
carried out using T7 and SP6 primers and the Sequenase DNA sequencing
kit (United States Biochemical, Cleveland, OH). DNA analysis and
alignment was done using the Genetics Computer Group sequence analysis
software package (Genetics Computer Group, Madison, WI).
Glutathione S-transferase fusion protein production,
purification, and antibody production. The 3'-end
of the rabbit skeletal muscle CNG channel gene was amplified by PCR
using primers 3128B and RACNG32 with an annealing temperature of
55°C and 2 min of extension at 72°C as described above. A
portion of this DNA from base 1914 (published aorta sequence numbering)
to the EcoR I site at base 2439 was
excised from pGEM and was cloned in frame into the vector pGEX-2T
(Pharmacia, Piscataway, NJ). This encoded a fusion of glutathione
S-transferase (GST) and the
COOH-terminal 145 amino acids of the rabbit aorta/muscle channel. This
construct was expressed in Escherichia
coli BL21. The bacteria were lysed, and the fusion
protein was purified on glutathione-Sepharose using the procedure of
Frangioni and Neel (11). The CNG channel peptide was eluted from the
resin with glutathione or by thrombin digestion (8 U/1 ml resin). The
purified, thrombin-eluted peptide was then used for commercial
production of polyclonal antiserum
rCNG3-1 in guinea pigs
(Cocalico Biologicals, Reamstown, PA).
Assembly of the entire gene and expression vector
production. Partial digestion with
EcoR I and digestion with
BamH I were used to isolate
complementary and nonoverlapping 5' and 3' pieces of the
CNG gene from the cloned skeletal muscle PCR products. The complete
coding sequence of the skeletal muscle CNG channel was assembled in the
BamH I site of pGEM by joining these
fragments at the EcoR I site at
position 1506 (aorta sequence numbering). The sequence of the assembled
coding region was determined by sequencing small restriction fragments
of this gene.
The vector pcDNA-rCNG, which uses a cytomegalovirus
promoter to drive high-level transcription in mammalian cells, was
produced by inserting the skeletal muscle CNG coding sequence into
pcDNA3 (Invitrogen, San Diego, CA). Digestion of the assembled muscle CNG gene with Eco47 III and
BamH I was used to isolate the region from bases 138 to 2669. This fragment was ligated into pcDNA3, which
had been digested with Kpn I, blunted,
and then digested with BamH I.
Cell culture and transfection. HEK
293T cells were grown in a humidified incubator with 5%
CO2 at 37°C in Dulbecco's
modified Eagle's medium supplemented with penicillin, streptomycin,
glutamine, and 10% fetal bovine serum. DNA for transfection was
isolated with the Qiagen plasmid MAXI kit (Qiagen). Transfections were performed with the Lipofectamine reagent (GIBCO-BRL) according to
manufacture's instructions. Cells were harvested 2-3 days
posttransfection.
Membrane isolation. Crude membranes
were isolated from HEK 293T cells using the procedure of
Coppi and Guidotti (5).
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline [10 mM
HEPES-tris(hydroxymethyl)aminomethane (Tris), pH 7.4, 140 mM NaCl,
and 5 mM KCl] was used in the place of phosphate-buffered saline.
The crude membranes were resuspended in hypotonic lysis buffer
containing 10 mM HEPES-Tris, pH 7.4, 50 mM sucrose, 2.5 µg/ml
aprotinin, and 1.0 µg/ml each of pepstatin A, chymostatin, and
leupeptin. For isolation of enriched plasma membranes, this membrane
suspension was loaded onto a sucrose step gradient containing two steps
of 15 and 30% sucrose in 10 mM HEPES-Tris, pH 7.4. The gradient was
centrifuged at 200,000 g at 4°C
for 2 h. Enriched plasma membranes were isolated from the 15:30%
sucrose interface. Membranes were diluted with lysis buffer and were
pelleted by centrifugation at 100,000 g at 4°C for 30 min. The membrane
pellets were subsequently resuspended in lysis buffer by
homogenization, frozen in a dry ice-acetone bath, and stored at
70°C.
Sarcolemma was isolated as described previously (28). These membranes
were washed by diluting the membranes two times in 1 M KCl and 10 mM
ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), pH 7.4, and pelleting the membranes at 100,000 g for 30 min at 4°C. The membrane
pellet was resuspended in lysis buffer by homogenization. Protein
concentration was determined on membranes solubilized in 1% sodium
dodecyl sulfate (SDS) with the Bio-Rad DC protein reagent (Bio-Rad,
Hercules, CA).
SDS-polyacrylamide gel electrophoresis and
immunoblotting. Membranes were solubilized in reducing
sample buffer (2% SDS, 65 mM Tris, pH 6.8, 5%
-mercaptoethanol)
and were separated by SDS-polyacrylamide gel electrophoresis (PAGE).
Column fractions were extracted with ether to remove lipid and were
precipitated with 6% trichloroacetic acid. The pellets were washed
with acetone and were resuspended in sample buffer before SDS-PAGE.
Proteins were transferred to nitrocellulose and were probed with
rCNG3-1 antiserum. Blots were probed with rabbit anti-guinea
pig immunoglobulin G-horseradish peroxidase (HRP) antiserum (Sigma) or
with protein A-HRP (Calbiochem, La Jolla, CA) and were developed with
the SuperSignal chemiluminescent substrate for Western Blotting
(Pierce, Rockford, IL) according to the manufacturer's instructions.
8-BrcGMP affinity chromatography and patch
clamping. Membranes prepared from HEK cells were
solubilized with CHAPS, purified by 8-BrcGMP affinity chromatography,
reconstituted, and prepared for patch clamping as described previously
(28) with the following exceptions. When preparing patch-clamp samples
from unsolubilized HEK membranes, only 0.1 µg of protein were used
per 100 µg of lipid. Patch clamping was performed in the absence of
EGTA because its presence tended to destabilize the patches. Inhibition
of CNG channel activity by µ-conotoxin GIIIB was tested by obtaining patches and assaying for CNG channel activity with the toxin already present in the pipette. This technique was used because, unlike sarcolemma, patches of transfected HEK membranes were not stable enough
to survive addition of the toxin to the pipette after patch formation
and demonstration of CNG channel activity.
Patch-clamp data were collected and analyzed with the program IGOR
(Wavemetrics, Lake Oswego, OR) as described previously (28). Titrations
were normalized to the plateau open current after subtraction of any
background current. Titrations were then fit by least squares to the
Hill equation
where
I is current,
Imax is maximum
current, [cGMP] is cGMP concentration, and n is
the Hill coefficient.
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RESULTS |
Cloning of the rabbit skeletal muscle CNG channel
coding sequence. To identify CNG channel genes
expressed in rabbit skeletal muscle, we used a PCR approach. The amino
acid sequence of the cyclic nucleotide binding domain of known CNG
channel genes was aligned, and two well-conserved stretches of this
region were identified. Degenerate oligonucleotide primers were
designed to correspond to the consensus sequence for these regions. As
can be seen in Fig. 1A, these
primers amplify a band of the expected size [174 base pairs
(bp)] from rabbit skeletal muscle cDNA but not from rabbit
skeletal muscle mRNA. This result indicates that a member of the CNG
channel family is expressed in rabbit skeletal muscle. The band
amplified from rabbit muscle cDNA was cloned and sequenced several
times and was shown to be identical to the CNG gene previously cloned
from rabbit aorta (Fig. 1B; see Ref. 1). The published sequence of this aorta gene (1) was therefore used to
design primers to clone the entire coding region from skeletal muscle
by PCR. All nucleotide and amino acid numbering used in this study is
that of the published aorta sequence (1). As shown in Fig.
1C, both the 5' (bases 99 to
1933)- and 3' (bases 1120 to 2669)-ends of the aorta CNG channel
gene can be amplified by PCR from skeletal muscle cDNA. These two
pieces cover the entire coding region of this gene (bases 153 to 2348)
and have an 800-bp overlap. An EcoR I
site (position 1506) in this overlapping region was used to join the
5'- and 3'-ends and to produce the entire skeletal muscle
CNG channel gene (aorta bases 99 to 2669). The entire skeletal muscle
gene was sequenced and is identical to the aorta sequence except for
two C to T transitions at positions 389 and 623. These changes do not
change the amino acid sequence and probably represent allelic variation
(data not shown).
PCR was also used to investigate the tissue distribution of expression
of the skeletal muscle gene. As shown in Fig.
2, a portion of this gene can be amplified
from skeletal muscle and stomach cDNA. Amplification from stomach may
indicate that this gene is expressed in smooth as well as skeletal
muscle. There may also be a low level of expression in kidney and brain
where faint bands can be seen in Fig. 2. Small amounts of additional bands were amplified from most tissues tested, probably due to nonspecific priming.
Expression of skeletal muscle CNG channel
gene. The COOH-terminal 145 amino acids of the skeletal
muscle CNG channel were expressed as a GST fusion and purified as
described in MATERIALS AND METHODS (data not shown). This
purified peptide was used for production of guinea pig antiserum
rCNG3-1 (produced by Cocalico Biologicals). As can be seen in
Fig. 3A, the preimmune
serum does not react with any proteins in the bacteria expressing the
fusion protein, whereas the immune serum reacts strongly with the
GST-CNG fusion. Only one additional band is visible in the bacterial
lysate.

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Fig. 3.
Immunoblotting with rCNG3-1 antiserum against the rabbit
skeletal muscle CNG channel COOH-terminus.
A: characterization of rCNG3-1
antiserum. Proteins were separated with 7.5% SDS-polyacrylamide gel
electrophoresis (PAGE) and transferred to nitrocellulose. Blot was
probed with a 1:1,000 dilution of preimmune serum and then washed and
reprobed with a 1:5,000 dilution of immune serum. Lane
1: bacterial lysate expressing glutathione
S-transferase (GST)-CNG fusion
protein; lane 2: purified GST-CNG
fusion protein. B: expression of the
muscle CNG channel protein. Plasma membranes were separated by 8%
SDS-PAGE and were probed with a 1:1,000 dilution of rCNG3-1.
Lane 1: untransfected HEK 293T cells
(10 µg); lane 2: HEK 293T
transfected with pcDNA-rCNG (10 µg); lane
3: rabbit sarcolemma (500 µg).
C: sarcolemma (375 µg) was blotted
as in B and was probed with preimmune
serum.
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The entire skeletal muscle CNG channel was expressed in tissue culture
cells by transient transfection. As can be seen in Fig.
3B, expression of the skeletal muscle
CNG channel gene leads to production of a 63-kDa protein that reacts
with the
rCNG3-1 antiserum. Rabbit sarcolemma also contains an
immunoreactive protein of the same size, confirming that this protein
is expressed in rabbit skeletal muscle (Fig.
3B, lane
3). This band is not seen when rabbit sarcolemma is
probed with preimmune serum (Fig.
3C). The dark band in sarcolemma at
42 kDa is the same size as actin and probably represents a nonspecific
reaction with this abundant muscle protein.
The properties of the muscle CNG channel were analyzed in HEK 293 cells
because these cells have previously been shown not to express any
endogenous CNG channel genes (7). As shown in Fig.
4, untransfected HEK cells do not show any
increased current with the addition of cGMP. In contrast, membranes
from cells transfected with the muscle CNG channel gene do show an
increase in current in response to the addition of increasing amounts
of cGMP, demonstrating that the product of the skeletal muscle CNG gene
does have CNG channel activity. Average currents of representative
patches containing untransfected or transfected membranes are shown in
Fig. 4A. Figure 4,
B and
C, shows individual trials of
experiments with transfected and untransfected membranes. When these
transfected membranes are patched in the presence of µ-conotoxin
GIIIB, an inhibitor of the native skeletal muscle CNG channel, this CNG
activity is inhibited (Fig. 4A).
Fitting of the cyclic nucleotide dependence of the expressed channel
with the Hill equation produces a fit with
K1/2 for cGMP of
6.15 ± 1.19 × 10
5
M and a Hill coefficient of 1.57 ± 0.53 (Fig.
5). These characteristics are most similar
to the low cGMP affinity form of CNG channel seen in rabbit skeletal
muscle (28).

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Fig. 4.
Expressed skeletal muscle protein has CNG channel activity. Plasma
membranes from transfected and untransfected HEK cells were used for
tip-dip patch clamping. Patches with gigaohm seals were picked up on
the end of a patch pipette, and current through the patches was tested
in the presence of increasing amounts of cGMP in the bath solution.
Current through the patches was measured at voltages from 80 to
+80 mV in 20-mV increments. Samples with toxin had 2 µM µ-conotoxin
GIIIB present in the bath and pipette throughout the experiment.
A: average current at +80 mV through
representative patches. Data are means + SE of at least 6 trials at
each cGMP concentration. Summary of the number of patches with CNG
channel activity is shown at bottom.
µ-Conotoxin data are from the only patch to show cGMP-dependent
current in the presence of toxin. B:
individual trials of untransfected plasma membranes. Current traces
were recorded in response to voltage pulses ranging from 80 to
+80 mV in 20-mV increments. C:
individual trials of transfected plasma membranes.
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Fig. 5.
Titration of expressed muscle CNG channel. Plasma membranes of
transfected HEK cells were tested by tip-dip patch clamping. Increasing
amounts of cGMP were added to the bath, and currents were recorded in
response to voltage pulses at +60 and +80 mV. Data are average currents
at +80 mV of individual titration experiments normalized to the plateau
open current. Each point is mean ± SE of the average current
recorded during 5 test voltage pulses. Data from all titration
experiments were fit by least squares to the Hill equation. Fit is
shown and has a half-maximal activation constant
(K1/2) for cGMP
of 6.15 ± 1.19 × 10 5 M and a Hill
coefficient of 1.57 ± 0.53. Imax, maximum
current; [cGMP], cGMP concentration.
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Isolation of CNG channels with 8-BrcGMP affinity
chromatography. Titrations of HEK membranes expressing
the muscle CNG channel gene seem to indicate that all CNG channel
activity in these cells is the low cGMP affinity channel form present
in skeletal muscle. However, the two forms of skeletal muscle CNG
channels only become apparent after solubilization and purification by
8-BrcGMP affinity chromatography (28). Therefore, we used isolation
with 8-BrcGMP affinity chromatography to investigate if both channel
forms seen in skeletal muscle are present in the transfected HEK cells.
As shown in Table 1, when transfected HEK
membranes are subjected to 8-BrcGMP affinity chromatography, CNG
channel activity elutes from the column in two populations that peak in
fraction E2 and in fraction E6 (Table 1). This elution profile is
strikingly similar to the pattern seen when rabbit sarcolemma is
purified by this method, where CNG channel activity also elutes from
the column in fractions E2 and E6 (28).
To further compare the two forms of CNG channel isolated from
transfected HEK cells with those present in skeletal muscle, we
investigated their cGMP dependence. Titration of the peak fractions of
CNG channel activity reveals that the HEK fraction E2 has a K1/2 for cGMP of
4.59 ± 0.95 × 10
5
M and a Hill coefficient of 1.37 ± 0.37, whereas HEK fraction E6
has a K1/2 of
5.27 ± 0.66 × 10
7 M and a Hill
coefficient of 2.91 ± 0.94 (Fig. 6).
These values are very similar to those seen in isolated skeletal muscle
CNG channel populations (fraction E2: K1/2 of
1.93 × 10
5 M and a Hill coefficient
of 1.16; fraction E6:
K1/2 of
5.79 × 10
7 M and
a Hill coefficient of 3.63; see Ref. 28). This suggests that the
single CNG channel gene cloned from skeletal muscle can produce
both of the channel forms that are present in skeletal muscle.

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Fig. 6.
cGMP titrations of isolated channel populations. Proteins from
fractions E2 and E6 of the 8-bromoguanosine 3',5'-cyclic
monophosphate (8-BrcGMP) column were reconstituted into liposomes and
patched by tip-dip patch clamping. Titrations were performed as in Fig.
5. Fit for fraction E2 has
K1/2 of 4.59 ± 0.95 × 10 5 M
and a Hill coefficient of 1.37 ± 0.37. Fit for fraction E6 has
K1/2 of 5.27 ± 0.66 × 10 7 M
and a Hill coefficient of 2.91 ± 0.94.
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Proteins from the 8-BrcGMP column fractions described in Table 1 were
precipitated, separated by SDS-PAGE, and immunoblotted to confirm that
the expressed channel protein is present in both isolated CNG channel
populations. The CNG channel band in the column fractions is diffuse
and streaked (Fig. 7). The rod CNG channel
behaves in a similar manner after purification by 8-BrcGMP affinity
chromatography (28). As can be seen in Fig. 7, the skeletal muscle CNG
channel protein also seems to elute from the 8-BrcGMP column in two
populations. The first population is in fractions E1 to E3, whereas the
second population is in fractions E5 and E6. This distribution
corresponds well to the distribution of the channel activity. The lower
band visible in the immunoblot was present in all lanes of this blot,
including the lane containing untransfected HEK 293T membranes (Fig.
7). These cells are known not to express any endogenous CNG channel
genes (7). Therefore, this lower band is a background band that is
unrelated to the skeletal muscle CNG channel.

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Fig. 7.
Immunoblot analysis of 8-BrcGMP column fractions. Column fractions were
precipitated with trichloroacetic acid and separated by SDS-PAGE as
described in MATERIALS AND METHODS. Blot was probed with
rCNG3-1 antiserum.
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DISCUSSION |
A gene for a CNG channel, which is identical to that previously cloned
from rabbit aorta (1), has been cloned from rabbit skeletal muscle. The
only sequence differences between the genes are two single base pair
changes that do not change the amino acid sequence of the protein.
However, there are differences between the characteristics of the two
expressed proteins. The product of the skeletal muscle gene in HEK 293T
cells shows a
K1/2 for cGMP of
6.15 ± 1.19 × 10
5
M and a Hill coefficient of 1.57 ± 0.53, whereas the product of the
aorta gene in oocytes has been reported to have a
K1/2 of 1.7 µM
and a Hill coefficient of 2.2 (1). This discrepancy may be due to the
different expression systems and patch-clamp techniques used to study
these genes. The aorta CNG channel was studied using injection of RNA
into Xenopus oocytes and inside-out patches excised from the oocytes. The skeletal muscle channel was
expressed by transient transfection into HEK cells and was tested using
tip-dip patch clamping of HEK plasma membranes.
The rabbit aorta/skeletal muscle gene is most similar to cloned
olfactory CNG channel genes (1). The only previous report of the
cloning of a CNG channel from skeletal muscle is from Feng et. al. (8),
who reported amplifying 200 bp of the rat rod CNG channel gene from
skeletal muscle. However, none of our PCR reactions on rabbit skeletal
muscle cDNA, including those using degenerate primers that would
amplify any known CNG channel gene, showed any evidence for the
expression of a rod channel in rabbit skeletal muscle. The primers used
by Feng et. al. (8) were specific for the rod CNG channel and therefore
would not have detected the expression of an olfactory CNG channel gene
in rat muscle. It has also previously been shown that the same
nonsensory tissue can express different CNG channel genes, depending on
the species (7).
Because the aorta/skeletal muscle CNG channel gene has an open reading
frame that encodes 732 amino acids, the expected product is a protein
of ~81 kDa. However, the protein detected in transfected tissue
culture cells and native skeletal muscle appears by SDS-PAGE to have a
mass of 63 kDa. The native rod
-subunit also has an apparent mass of
63 kDa on SDS-PAGE, although its gene encodes a protein with a
theoretical molecular weight of 79,000 (4, 19). This discrepancy has
been attributed to the proteolytic removal of the
NH2-terminal 92 amino acids,
producing a protein with a theoretical molecular weight of 69,000 (24).
A similar cleavage might reduce the size of the skeletal muscle CNG
protein. Alternatively, the size difference may be due to the use of a different ATG to initiate translation. The aorta CNG channel start site
of translation was assigned to the first in frame ATG, which occurs
after a stop codon, at position 153 (1). However, because this codon is
not in a perfect consensus sequence for initiation of translation, it
has been suggested that the actual start site might be the third ATG,
at position 357 (1). The codon at position 357 is in a better consensus
sequence and is homologous to the ATG used to initiate translation of
the olfactory CNG channel (1). If translation does initiate at position
357 then the theoretical molecular weight of the aorta/skeletal muscle
CNG protein would be 73,000, much closer to the observed size of the protein. Any remaining discrepancies in size might be due solely to the
difficulty in determining the size of membrane glycoproteins by
SDS-PAGE.
Expression of the skeletal muscle CNG channel gene in HEK cells
followed by purification with 8-BrcGMP affinity chromatography produces
two populations of CNG channel activity that are very similar to the
two forms seen after purification of native skeletal muscle CNG
channels. Both the native and expressed channels elute from the column
in two distinct populations, in fractions E2 and E6. The cyclic
nucleotide dependences of the native and expressed populations are also
strikingly similar. Fraction E2 from native skeletal muscle has a
K1/2 for cGMP of
1.93 × 10
5 M, whereas
that from HEK plasma membrane has a
K1/2 of 4.59 × 10
5 M. The native
and expressed E6 populations are even more similar with
K1/2 values of
5.79 × 10
7 M and
5.27 × 10
7
M, respectively (for native values see Ref. 28). Because the expressed
channel in HEK membranes appears to be primarily in the low-affinity
state, it might be expected that there would be more activity in the E2
fraction than in the E6 fraction after affinity chromatography. This
doesn't seem to be the case, probably because the low-affinity form is
less tightly bound to the column. Immunoblotting reveals that the
expressed skeletal muscle CNG channel protein also elutes from the
column in two populations. These results strongly suggest that the two
CNG channel forms present in skeletal muscle can be explained by the
expression of a single CNG channel gene, the aorta gene.
The finding that expression of one CNG channel gene can produce
channels with widely varying cGMP affinities is not entirely unprecedented. It is well documented that the apparent affinity of the
CNG channels can be modulated over a wide range. For example, phosphorylation of or nickel binding by the rod channel can change its
apparent cGMP affinity by an order of magnitude (13, 14). Additionally,
covalent cross-linking of cGMP analogs to the native rod CNG channel
has demonstrated two populations of cGMP binding sites with differing
cGMP affinities. Cross-linking of cGMP to the rod
-subunit expressed
in Xenopus oocytes also demonstrated that these channels can have two populations of cGMP binding sites (17). The affinities of the two populations of cGMP binding sites seen
by cross-linking, with
K1/2 values of
0.42 and 16 µM, are similar to those of isolated skeletal muscle
channels (17, 28).
The greatest difference between the native and expressed skeletal
muscle CNG channels is the cyclic nucleotide dependence of the channels
present in unsolubilized membranes. The CNG channels present in
unsolubilized skeletal muscle membranes are most similar to the high
cGMP affinity form that is seen after 8-BrcGMP affinity chromatography
(28). The expressed CNG channels in HEK plasma membranes, on the other
hand, are most similar to the low cGMP affinity form. This finding
supports the idea that the low-affinity form of skeletal muscle CNG
channel is not an artifact of denaturation during solubilization.
Isolation of the CNG channels by 8-BrcGMP affinity chromatography
demonstrates that both high and low cGMP affinity channels are present
in HEK and skeletal muscle membranes. Perhaps a ubiquitous modification
system modulates the affinity of the skeletal muscle channel, and the
levels of this modulation are controlled in a tissue-specific manner.
Despite these differences, both the native skeletal muscle CNG channel,
which is responsible for insulin-activated sodium entry, and the
expressed CNG channel can be inhibited by µ-conotoxin GIIIB.
The fact that expression of one CNG channel protein can produce both
forms of skeletal muscle CNG channels could have important implications
for insulin-activated sodium entry. Insulin may increase sodium entry
by increasing cGMP levels and thereby opening the CNG channels. Most of
the native skeletal muscle channels may be in the high-affinity form to
make them sensitive to small changes in a low level of cGMP.
Alternatively, insulin could act to increase the percentage of channels
in the high-affinity form, which would increase sodium entry at resting
cGMP levels. No matter what the action of insulin, the fact that a
single CNG channel protein can produce two CNG channel forms with such
disparate cGMP affinity confirms that these channels are not the static
sensors of cGMP levels, as was thought when they were first discovered.
CNG channels can be modulated by many signals and can produce channels
with a large range of affinities. This provides cells expressing these channels with great flexibility in controlling cation entry and in
linking this entry to signaling systems.
We thank Dr. Maddalena Coppi for reading the manuscript and
providing insightful comments.
This work was supported by National Institutes of Health Grants
DK-27626 and GM-07598.
Address for reprint requests: L. C. Santy, Casanova Lab, Pediatric
Gastroenterology and Nutrition, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129.