(Received for publication, September 13, 1995; and in revised form, November 21, 1995)
From the
The cDNA of three variants of a cyclic nucleotide-gated (CNG)
channel modulatory subunit (CNG4c-CNG4e) has been cloned. CNG4c,
CNG4d, and CNG4e differ slightly from each other within an
amino-terminal sequence that was originally reported as part of the
bovine retinal glutamic acid-rich protein (GARP). The core region of
CNG4 is homologous to the second subunit of the human rod photoreceptor
channel (hRCNC2b), suggesting that both proteins are alternatively
spliced products of the bovine and human homologue of the same gene.
CNG4 transcripts are present in retina, testis, kidney, heart, and
brain. Expression of CNG4 in HEK293 cells did not lead to detectable
currents. Coexpression of CNG4 with the principal subunit of the bovine
testis CNG channel (CNG3) resulted in currents which differed in
several aspects from that induced by CNG3 alone. The heterooligomeric
CNG3/CNG4 and the homooligomeric CNG3 channels were modified by
Ca-calmodulin and some calmodulin antagonists. The
results suggest that CNG4 forms functional heterooligomeric channels
with CNG3 in vitro and probably also in intact tissues.
Cyclic nucleotide-gated (CNG) ()channels comprise a
class of nonselective cation channels that are directly and
cooperatively activated by the binding of cGMP or
cAMP(1, 2) . Originally, CNG channels have been
characterized in sensory cells by electrophysiological techniques and
molecular cloning(3, 4) . In vertebrates, three
different genes encode CNG channels. CNG1 is expressed in rod
photoreceptors(5, 6, 7, 8) , CNG3 in
cone photoreceptors(8, 9) , and CNG2 in olfactory
neurons(10, 11, 12) . Recently, an additional
CNG channel has been identified in Drosophila, which is
expressed both in photoreceptors and olfactory sensillae(13) .
There is solid evidence that expression of CNG channels is not
restricted to sensory cells. Electrophysiologically, cGMP-activated
cation channels have been detected in photosensitive cells of pineal
glands(14) , in retinal bipolar
cells(15, 16) , and in some retinal ganglion cells (17) . PCR amplification has identified partial sequences of
CNG1-3 channels in several tissues and
species(17, 18, 19) . In addition, a
functional olfactory-like channel (rACNG) has been cloned from rabbit
aorta(20) . Furthermore, mammalian CNG3 was originally cloned
from bovine kidney (21) and testis(9) .
Additional
subunits (referred to as subunit or subunit 2) have been cloned
from the rod outer segment (hRCNC2a and hRCNC2b, (22) ) and
from olfactory neurons (rOCNC2, (23) and (24) ). These
new subunits do not form functional channels by themself. They
coassamble with CNG1 and CNG2 to form heterooligomeric complexes and
induce channel properties that are present in the native channels but
are absent from the channels formed by the CNG1 and CNG2 homooligomers (22, 23, 24) . The ``new'' but
physiological properties include activation of the channel by cAMP at
physiologically observed concentrations, single-channel flickering, and
block of the current by L-cis-diltiazem. It was
unknown whether or not the CNG3 channel expressed in sensory or
nonsensory cells is also coassembled with a modulatory subunit. Here,
we report the cloning of three variants of a cDNA encoding an
additional CNG channel subunit that modulates the functional properties
of the CNG3 channel.
Figure 1: Primary structure of bovine testis CNG4c-CNG4e. A, scheme of CNG4c. The open box represents the amino acid sequence with the amino terminus on the left and the carboxyl terminus at the right. Putative transmembrane domains(1, 2, 3, 4, 5, 6) , the pore region (P), and the binding site for cGMP are indicated. The sequence common with GARP is shown as a gray box. B, nucleotide sequence, deduced amino acid sequence of CNG4c, and its alignment with subunit 2 of human retinal rod cGMP-gated cation channel (hRCNC2b). The nucleotide sequence was assembled from clones pRACE12, pcGT7, and pcGT4. Amino acids identical between CNG4c and hRCNC2b are boxed. Gaps in the sequence of hRCNC2b are represented by dashes. At positions 15 and 750 of hRCNC2b stretches of 15 and 2 glutamic acid residues, respectively, are indicated by indices. The putative transmembrane domains, the pore region, and the cGMP binding site are shown by lines below the amino acid sequences. The sequence missing in CNG4d and CNG4e is illustrated by black and gray boxes, respectively. The stop codons preceding the initiation ATG and at the end of the open reading frame of CNG4c, respectively, are double-underlined. The sequence of CNG4c, which is also present in GARP, is marked by arrows. The sequences flanking the 5` and 3` end of the common region in GARP are shown in minuscule over the nucleotide sequence of CNG4c. Three nucleotide exchanges with respect to the published sequence of GARP, which were found both in GARP itself and CNG4c, are also shown in minuscule over the sequence.
Figure 2:
Tissue expression of CNG3, CNG4, and GARP
transcripts. A, scheme showing the location of probes A-C used
for Northern analysis. The protein coding regions of GARP and CNG4c
sequences are represented as interrupted gray and white boxes,
respectively. Only the carboxyl-terminal part of GARP and the
amino-terminal part of CNG4c is shown. 5`- and 3`-untranslated
sequences (5`UTR and 3`UTR) are shown as horizontal lines. The sequence common to both proteins is
indicated in black. Probes A-C are shown as black lines below the proteins. B, Northern analysis
of CNG4 and GARP expression. Ten micrograms of poly(A)
RNA of bovine testis and retina RNA was used in each lane. The same
blot was subsequently hybridized with probes A, B, and C as described
under ``Experimental Procedures.'' Autoradiographic exposure
at -70 °C was 30 h for testis and 3 h for retina. With probe
C no signal was seen in testis even after an exposure for 5 days. C, PCR amplification of CNG3- and CNG4-specific sequences. For
each tissue two separate PCR reactions were performed using either
CNG3- or CNG4-specific primer pairs as described under
``Experimental Procedures.'' One-tenth of a CNG3- and
CNG4-specific amplification reaction was combined and separated on a 5%
polyacrylamide gel. The identity of the amplified sequences was
ascertained by sequencing of the respective
bands.
The sequence from residues
116-939 of CNG4c is highly homologous to the sequence of hRCNC2b.
By contrast, the NH-terminal end of CNG4c shows only minor
homology to hRCNC2b. The major part of this divergent sequence
(residues 10-115) is present in a 65-kDa glutamic acid-rich
protein (GARP) that has been cloned from bovine rod outer
segment(25) . Within the common region of 320 bp the cDNAs of
CNG4c and GARP differ only at three positions. These differences most
likely reflect an individual sequence polymorphism since they were also
present in a partial GARP sequence amplified from bovine testis mRNA
with GARP-specific primers (data not shown). The deletions present in
CNG4d and CNG4e are localized within the 320-bp GARP sequence. PCR
analysis indicated that at least the 27-bp deletion giving rise to
CNG4d is also represented in the GARP-specific cDNA (data not shown).
Northern analysis with probes A and B identified CNG4 transcripts only in testis and retina. To detect possible low level expression of channel subunits, we performed PCR reactions with degenerate primers deduced from the core regions of CNG3 and CNG4. Specific fragments were obtained from the cDNA of several bovine and rat tissues (Fig. 2C). The sequences of rat CNG3 and CNG4 are highly homologous to their bovine counterparts with nucleotide sequence identities of 89 and 90%, respectively. CNG3 could be detected in bovine retina, kidney, and heart as well as in rat kidney, heart, and brain being in line with previous results(21) . By contrast, CNG4 was present in all rat tissues tested but was absent from bovine kidney and heart mRNA.
The
cGMP-induced current of the heterooligomeric CNG3/CNG4d channel was
blocked by L-cis-diltiazem with a half-maximal
inhibition (IC) of 10 µM (Fig. 3A), whereas the homooligomeric CNG3 channel
was affected only marginally by 100 µML-cis-diltiazem (Fig. 3B). The
IC
value of the heterooligomeric channel is in the range
of IC
values reported for native cone
photoreceptors(30) , whereas the native (31) and the
heterologously expressed rod channel (22) is about 10 times
more sensitive for L-cis-diltiazem. Like the native
rod and cone channels the CNG3/CNG4d channel was blocked by L-cis-diltiazem in a time- and voltage-dependent
manner (Fig. 3C). The block was more effective at
positive than at negative membrane potentials (Fig. 3C).
Figure 3:
The
heterooligomeric CNG3/CNG4 channel and the homooligomeric CNG3 channel
differ in their sensitivity to L-cis-diltiazem and
their single-channel properties. A and B, current
traces of the CNG3/CNG4d (A) and the CNG3 channel (B)
induced at +60 mV by 0.3 mM cGMP in the absence and
presence of L-cis-diltiazem (Dilt).
Ca-free Ringer solution was used in both bath and
pipette. C, steady-state current-voltage relations of the
CNG3/CNG4d current induced by 0.3 mM cGMP in the absence
(
) or presence (
) of 10 µML-cis-diltiazem. The inset shows
current traces induced at ±60 mV in the absence and the presence
of 10 µM diltiazem to illustrate the characteristic time
dependence of the L-cis-diltiazem-induced current
block.
, current-voltage relation in the absence of cGMP. D, single-channel currents of CNG3 and CNG3/CNG4d channels
induced by 1 µM cGMP.
CNG4d weakened the outward rectification
observed in the presence of 2 mM extracellular Ca with the homooligomeric channel (data not shown) and induced
single-channel flickering (Fig. 3D). Furthermore, CNG4d
increased the affinity of the channel for cyclic nucleotides (Table 1). The increase in affinity was more prominent with cAMP
(6-fold decrease of K
at +60 mV) than with
cGMP (1.5 fold decrease of K
). Interestingly, the
high degree of cooperativity found for the activation of the
homooligomeric CNG channel by cAMP (apparent Hill coefficient of over
4) was decreased when CNG4d was present (apparent Hill coefficient of
1.5).
Figure 4:
Inhibitory effect of
Ca-calmodulin on the heterooligomeric CNG3/CNG4d
channel and the homooligomeric CNG3 channel. The top panels show current traces induced by 20 µM cGMP in 50
µM free Ca
solution at a voltage range
of ±90 mV with voltage increments of ±30 mV. The traces
are corrected for currents elicited in the absence of ligand. The bottom panels show current traces recorded 1 min after
application of 50 µM free Ca
+ 230
nM calmodulin (CaM) to the same
patches.
Figure 5:
Potentiation of the inhibitory effect of
channel blockers by Ca-calmodulin. A,
CNG3/CNG4d; B, CNG3. The left panels represent the
current-voltage relation of individual patches perfused by 50
µM free Ca
-solution in the absence of
cGMP (
), followed by 0.3 mM cGMP (
), followed by
0.3 mM cGMP plus the indicated channel blocker (
) and
followed by cGMP plus channel blocker plus 230 nM calmodulin
(
). Dilt, 10 µML-cis-diltiazem; Calm, 3 µM calmidazolium; Pimo, 10 µM pimozide. The
steady-state effects of blockers and calmodulin were taken 30 s after
application of blockers or 60 s after coapplication of blocker +
calmodulin. The right panels show the extent of block in the
absence (&cjs2113;) and presence of 230 nM calmodulin (
)
at ±60 mV.
, currents in the absence of calmodulin and
blocker. The values for current block (number of experiments/percent of
normalized current at -60 mV/% of normalized current at +60
mV) were as follows. CNG3+CNG4d: L-cis-diltiazem
(8/64.6 ± 4.3/28.4 ± 2.9), L-cis-diltiazem +
Ca
-calmodulin (8/27.4 ± 5.6/7.9 ± 1.1),
calmidazolium (10/57.7 ± 7.7/58.2 ± 7.6); calmidazolium
+ Ca
-calmodulin (10/12.3 ± 4.6/10.5
± 3.6), pimozide (8/91.4 ± 7.5/88.2 ± 7.2),
pimozide + Ca
-calmodulin (8/43.2 ±
8.9/38.2 ± 8.6). CNG3: L-cis-diltiazem (7/87.7
± 3.2/97.3 ± 4.4), L-cis-diltiazem
+ Ca
-calmodulin (7/13.9 ± 5.4/17.6
± 4.7), pimozide (6/83.1 ± 4.9/81.4 ± 5.1),
pimozide + Ca
-calmodulin (6/35.0 ±
9.6/30.3 ± 9.7).
The same experiments were
repeated with the homooligomeric CNG3 channel. Pimozide blocked the
CNG3-mediated current to the same extent as that through the
heterooligomeric CNG3/CNG4d channel (Fig. 5B). L-cis-Diltiazem reduced the current through the CNG3
channel only by 5% at +60 mV in the absence of calmodulin.
However, 10 µML-cis-diltiazem blocked
the current through the CNG3 channel by 83% in the presence of
Ca-calmodulin (Fig. 5B). This block
was quantitatively similar to the block of the CNG3/CNG4d channel by L-cis-diltiazem. In contrast to the heterooligomeric
channel, the block of CNG3 by L-cis-diltiazem was not
voltage-dependent, indicating that CNG4d was responsible for the
voltage dependence of the block of the heterooligomeric channel.
Three splice variants of a modulatory subunit of the
CNG3 channel, CNG4c, CNG4d, and CNG4e, have been cloned from bovine
testis. The COOH-terminal part of the CNG4 sequence (residues
116-939), which contains the transmembrane segments, the pore
region, and the cGMP binding domain, is homologous to the second
subunit of the human rod photoreceptor channel hRCNC2b(22) . By
contrast, the NH
-terminal part (residues 1-115)
reveals only weak homology to the sequence of hRCNC2b but contains a
sequence (residues 10-115) that is essentially identical with a
COOH-terminal portion of the retinal 65-kDa glutamic acid-rich protein
(GARP, (25) ). The first nine residues of CNG4 are unique to
this protein and show no homology to either GARP or hRCNC2b. The
partial sequence identity between GARP and CNG4 suggests that both
proteins are derived from the same gene either by alternative splicing
or by transcription from different promotors.
Northern analysis detected CNG4-specific transcripts of different size in testis (3.3 and 3.5 kb) and retina (7.5 kb). These transcripts might represent alternatively spliced variants of CNG4 expressed in these tissues. The full-length GARP is expressed in significant amounts only in retina. At present, the function of GARP is not known, but it is tempting to speculate that GARP and CNG4 modulate the same function in retina. CNG4-specific mRNA was detected in small amounts in several other tissues. Interestingly, bovine heart and kidney did not contain significant amounts of CNG4 transcripts, suggesting that the CNG3 channels present in these tissues are either homooligomeric channels or contain another modulatory subunit which has not been identified so far.
Similar to subunits of other CNG
channels(22, 23, 24) , each of the three CNG4
variants modulated the properties of the CNG3 channel, but failed to
induce cGMP-dependent currents when expressed alone in HEK 293 cells.
The modulated properties included a shift in the apparent K
values for cGMP and cAMP, single-channel
flickering, and a sensitive block by L-cis-diltiazem
in the absence of calmodulin. The IC
value of 10
µM and the time and the voltage dependence of the L-cis-diltiazem block were indistinguishably from
that reported for the native cone channel (30) . CNG4 did not
change substantially the current-voltage relation in the absence of
divalent cations. By contrast, it decreased the outward rectification
of the current observed in the presence of extracellular
Ca
, indicating that CNG4 influenced the voltage
dependence of the block. These results strongly support the notion that
CNG4 and CNG3 form heterooligomeric channels in HEK cells and in intact
tissues.
The activation of both homooligomeric and heterooligomeric
channels is modulated by Ca-calmodulin. At
physiological concentrations, Ca
-calmodulin increased
about 2-3-fold the K
value for cGMP in both
channels. The increase is qualitatively similar to the
Ca
-calmodulin-dependent increase observed in native
rod photoreceptor channels(32) . However, in rod photoreceptor
channels the effects of calmodulin required the presence of the long
version of the second subunit hRCNC2b(29) . Calmodulin had no
effect on the homooligomeric CNG1 channel. The direct inhibition of
CNG3 activation resembles the direct effect of
Ca
-calmodulin on the olfactory ion conducting CNG2
subunit(27, 36) , although calmodulin shifted the
apparent K
values only by 2-3-fold compared
with a 10-20-fold shift in the olfactory channel. In support of
these electrophysiological results, CNG3, but not CNG1, contains a
sequence in the amino terminus that is similar to the
Ca
-calmodulin binding region identified in
CNG2(36) .
The major effect of calmodulin observed was the
potentiation of channel block at saturating cGMP concentrations. L-cis-Diltiazem, pimozide, and calmidazolium, which
are also known to block native olfactory and photoreceptor
channels(33, 34, 35) , blocked the homo- and
heterooligomeric channels. Most strikingly, the current induced by CNG3
alone became sensitive to L-cis-diltiazem in the
presence of Ca-calmodulin. In contrast to the
heterooligomeric channel, the extent of the block of CNG3 by L-cis-diltiazem was almost identical at negative and
positive membrane potentials, indicating that CNG4 was responsible for
the voltage-dependent L-cis-diltiazem block. The
molecular basis of the observed calmodulin-dependent potentiation is
unclear, but probably involves a conformational change of the channel
protein induced by Ca
-calmodulin that increases the
affinity of the channel for the used compounds.
The expression of two CNG channel subunits in bovine testis, which together form a functional channel, strongly supports the notion that heterooligomeric CNG channels are not restricted to sensory cells. CNG4 transcripts were not identified in all tissues containing CNG3 mRNA. It is therefore likely that CNG3 forms homooligomeric channels in some cells as has been proposed for the olfactory (23) and the rod photoreceptor channel(37) . Future investigations will be necessary to study the functional implications of these differences in subunit composition.
After submission of this manuscript the cloning and
functional expression of the full-length subunit (bRCNGC
)
from bovine rod outer segment has been reported(38) .
bRCNGC
is identical with the calmodulin binding 240-kDa protein
that copurifies with the rod photoreceptor CNG1 channel
protein(39, 40, 41) . Both CNG4 and
bRCNGC
have a bipartite structure consisting of a COOH-terminal
`-part, which is homologous to hRCNC2b, and a NH
terminus, which is homologous to GARP. Whereas CNG4 contains only
a small portion of GARP (residues 466-571), almost the complete
GARP sequence (residue 1-571) is present in bRCNGC
. In
addition, bRCNGC
lacks the NH
-terminal nine amino
acids of CNG4. Within the common region CNG4 and bRCNGC
differ at
five positions: residues 26 (Gln), 827 (Ala), 833 (Ala), and 880 (Glu)
are replaced in bRCNGC
by Arg, Ser, Arg, and Asp, respectively,
and residue 881 (Ala) is missing in bRCNGC
. It is likely, that
CNG4 and bRCNGC
are splice variants of the same primary
transcript. CNG4c and bRCNGC
have been expressed together with the
CNG3 and CNG1 channels, respectively. These
subunits modulate
several properties of the cGMP-induced current (i.e. single
channel flickering, sensitivity to L-cis-diltiazem)
in a comparable manner, being consistent with the finding (38) that the GARP part of bRCNGC
has no major influence
on the electrophysiological properties of the rod CNG channel. However,
the effect of the
subunit might depend on the type of
subunit which is coexpressed. Unlike the CNG1 channel, the
homooligomeric CNG3 channel is inhibited by calmodulin in the absence
of a
subunit. Coexpression of CNG4 does not induce an additional
effect. For this reason it cannot be decided from our experiments
whether CNG4 is like bRCNGC
a target for calmodulin. Further
experiments will be necessary to identify the location of the
calmodulin binding site.