From the Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rod photoreceptor cyclic nucleotide-gated (CNG) channels are modulated by tyrosine phosphorylation. Rod CNG channels expressed in Xenopus oocytes are associated with constitutively active protein tyrosine kinases (PTKs) and protein tyrosine phosphatases that decrease and increase, respectively, the apparent affinity of the channels for cGMP. Here, we examine the effects of genistein, a competitive inhibitor of the ATP binding site, on PTKs. Like other PTK inhibitors (lavendustin A and erbstatin), cytoplasmic application of genistein prevents changes in the cGMP sensitivity that are attributable to tyrosine phosphorylation of the CNG channels. However, unlike these other inhibitors, genistein also slows the activation kinetics and reduces the maximal current through CNG channels at saturating cGMP. These effects occur in the absence of ATP, indicating that they do not involve inhibition of a phosphorylation event, but rather involve an allosteric effect of genistein on CNG channel gating. This could result from direct binding of genistein to the channel; however, the time course of inhibition is surprisingly slow (>30 s), raising the possibility that genistein exerts its effects indirectly. In support of this hypothesis, we find that ligands that selectively bind to PTKs without directly binding to the CNG channel can nonetheless decrease the effect of genistein. Thus, ATP and a nonhydrolyzable ATP derivative competitively inhibit the effect of genistein on the channel. Moreover, erbstatin, an inhibitor of PTKs, can noncompetitively inhibit the effect of genistein. Taken together, these results suggest that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel catalyzed by PTKs, genistein triggers a noncatalytic interaction between the PTK and the channel that allosterically inhibits gating.
Key words: cyclic guanosine 5'-monophosphate; protein kinase; rod outer segment; photoreceptor; phosphorylation ![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyclic nucleotide-gated (CNG)1 channels play a central role in both visual and olfactory sensory transduction. CNG channels are directly activated by the binding of cyclic nucleotides to the channel protein. Recent
studies show that the sensitivity of CNG channels to cyclic nucleotides is not fixed, but rather can be modulated by other cellular factors. Transition metals (Ildefonse and Bennett, 1991; Karpen et al., 1993
; Gordon
and Zagotta, 1995
), diacylglycerol analogues (Gordon
et al., 1995b
), and the local anesthetic tetracaine
(Fodor et al., 1997a
,b) all bind to rod photoreceptor
CNG channels and alter their apparent affinity for
cGMP. In addition, Ca2+ modulates the sensitivity of
CNG channels, but it acts in an indirect manner. Thus,
Ca2+ decreases the apparent affinity for cGMP (increases the K1/2 value) by binding to calmodulin and
other unidentified Ca2+-binding proteins, which then
interact with the CNG channel protein (Hsu and Molday, 1993
; Chen et al., 1994
; Gordon et al., 1995a
).
We have recently shown that rod CNG channels can
also be modulated by tyrosine phosphorylation (Molokanova et al., 1997). Rod CNG channels expressed in
Xenopus oocytes exhibit a spontaneous increase in
cGMP sensitivity after patch excision, and this is reversed by application of ATP. These changes in cGMP
sensitivity are blocked by specific inhibitors of protein
tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs), respectively. These results imply that the
channel is associated with PTKs and PTPs that remain
active for many minutes after patch excision. Additional studies (Molokanova Maddox, Luetje, and
Kramer, manuscript submitted for publication) show
that mutagenesis of a specific tyrosine in the
subunit
of the rod CNG channel greatly reduces modulation, suggesting that the crucial phosphorylation site is located in the channel protein itself.
In this paper, we study the effects on CNG channels
of genistein, a broad-spectrum PTK inhibitor isolated
from legumes (Akiyama et al., 1987). PTKs have a conserved binding site for ATP and an additional distinct
site for binding of their protein substrate (Ullrich and
Schlessinger, 1990
). Genistein is a competitive inhibitor with respect to ATP in the kinase reaction and a
noncompetitive inhibitor with respect to the peptide
substrate, suggesting that genistein specifically interacts
with the ATP-binding site. Several other proteins that
possess ATP-binding sites are similarly influenced by
genistein. Thus, genistein competes for ATP-binding
sites on histidine kinase (Huang et al., 1992
) and topoisomerase II (Markovits et al., 1989
), inhibiting these
enzymes, and on the cystic fibrosis transmembrane conductance regulator, potentiating activation of this ion
channel (Weinreich et al., 1997
; Wang et al., 1998
).
This paper shows that genistein inhibits the rod
CNG channel, above and beyond its inhibitory effect
on tyrosine phosphorylation. The simplest explanation
for this inhibition would involve a direct binding of
genistein to the CNG channel. However, unlike all of the established direct targets for genistein action, CNG
channels do not appear to contain ATP binding sites.
Examination of the amino acid sequence of the rod
channel subunit does not reveal conserved ATP-binding domains (Kaupp et al., 1989
), and the only known physiological effects of ATP on CNG channels
occur through its participation in phosphorylation reactions (Molokanova et al., 1997
). Hence, we have considered the possibility that genistein does not bind
directly to the channel, but rather acts indirectly by
binding to an accessory protein that then binds to the
CNG channel. Since our previous studies indicate that
the expressed CNG channel is closely associated with
PTKs, we considered the possibility that genistein inhibition involves a noncatalytic effect of the PTK. Remarkably, we observe that the effect of genistein on
the channel is suppressed by erbstatin, another PTK
inhibitor, and by a nonhydrolyzable ATP analogue,
suggesting that the receptor for genistein that mediates inhibition of the rod CNG channel is indeed a
PTK. Hence, we propose that PTKs affect rod CNG
channels in two ways: (a) by allosterically regulating
channel gating, and (b) by catalyzing phosphorylation
of the channel protein.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression and Recording from Rod CNG Channels Expressed in Xenopus Oocytes
A cDNA clone encoding the bovine rod photoreceptor CNG
channel subunit (Kaupp et al., 1989
) was used for in vitro transcription of mRNA, which was injected into Xenopus oocytes (50 nl
per oocyte at 1 ng/nl). After 2-7 d, the vitelline membrane was
removed from injected oocytes, which were then placed in a
chamber for patch clamp recording at 21-24°C. Glass patch pipettes (2-3 M
) were filled with a solution containing 115 mM
NaCl, 5 mM EGTA, 1 mM EDTA, and 10 mM HEPES, pH 7.5, NaOH. This also served as the standard bath solution and cGMP
perfusion solution. EDTA was not included in solutions containing the Mg2+ salt of ATP or its analogues. After formation of a gigaohm seal, inside-out patches were excised and the patch pipette was quickly (<30 s) placed in the outlet of a 1-mm diameter tube for application of cGMP and other agents. We used a
perfusion manifold containing up to eight different solutions
that is capable of solution changes within 50 ms. Most patches
contained 100-200 channels. To calculate the apparent affinity
of CNG channels to cGMP, a series of four to five cGMP concentrations was applied to the patch. Steady state cGMP-activated
currents were normalized to saturating responses and fit to the
Hill equation to determine the apparent affinity (K1/2 value) for
cGMP.
PTK inhibitors (genistein, erbstatin, lavendustin A) were prepared as concentrated stock solutions in water or DMSO, and aqueous solutions containing the final concentrations were prepared for use as needed. The final concentration of DMSO did not exceed 0.1%, which had no effect on CNG channels or their modulation. Cyclic GMP, ATP, and AMP-PNP were purchased from Sigma Chemical Co., genistein, daidzein, erbstatin (stable analogue), and lavendustin A were purchased from Alexis Corp.
Current responses through CNG channels were obtained with
an Axopatch 200A patch clamp (Axon Instruments), digitized,
stored, and later analyzed on a 486 PC using pClamp 6.0 software. Membrane potential was held at 75 mV in all experiments. Current responses were normalized to the maximal CNG
current (Imax), elicited by saturating (2 mM) cGMP. Normalized
dose-response curves were fit to the Hill equation: I/Imax = 1/
[1+(K1/2 /A)N], using a nonlinear least squares fitting routine
(Origin; Microcal Software, Inc.), where A is the cGMP concentration and N is the Hill coefficient. To estimate the Ki for
genistein, we used a modified Hill equation: Ib/Imax = [1
(Ib(max)/Imax)]/[1 + (Ki/B)N] + Ib(max)/Imax, where B is the concentration of blocker, Ib and Ib(max) are the currents activated by
saturating cGMP in the presence of a given blocker concentration, and a saturating blocker concentration, respectively. Variability among measurements is expressed as mean ± SEM.
Recording from CNG Channels from Rod Outer Segments
Water-phase tiger salamanders (Ambystoma tigrinum) maintained
in a temperature-controlled aquarium (16°C) on a 12/12 light/ dark cycle were used in all experiments at the same time of day. Animals were dark adapted for 1 h and anesthetized in an ice-cold solution containing 1 g/liter of 2-amino benzoic acid for 20 min before decapitation and removal of eyes under dim red
light. Eyes were placed in saline containing (mM) 155 NaCl, 2.5 KCl, 1 CaCl2, 2 MgCl2, 10 glucose, and 10 HEPES, pH.7.5. Retinas were removed and gently triturated to yield isolated rods or
rod outer segments. Borosilicate glass pipettes (3-5 M) were
filled with standard patch solution (see above) and used to obtain excised inside-out patches from the outer segment.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of Genistein and Other PTK Inhibitors on CNG Channels
Rod CNG channels expressed in Xenopus oocytes are
modulated by changes in tyrosine phosphorylation state,
catalyzed by PTKs and PTPs that are native to the oocyte
membrane (Molokanova et al., 1997). Phosphorylation
lowers the cGMP sensitivity of the channel, whereas dephosphorylation increases cGMP sensitivity. Hence, after a membrane patch is excised from the oocyte, the channels gradually become dephosphorylated, resulting in a dramatic increase in cGMP sensitivity (decrease
in K1/2). When patches are excised into a solution that
contains ATP, there is little spontaneous change in
cGMP sensitivity, apparently because dephosphorylation by tyrosine phosphatases is balanced by phosphorylation by PTKs. We have shown that addition of PTK inhibitors blocks phosphorylation and shifts this equilibrium towards dephosphorylation, resulting in an increase in sensitivity to cGMP (Molokanova et al., 1997
).
To learn more about modulation of the rod CNG channel by PTKs, we examined the effects of genistein, a broad-spectrum PTK inhibitor. Fig. 1 A shows that genistein, like two other PTK inhibitors (lavendustin A and erbstatin), causes an increase in cGMP sensitivity (decrease in K1/2) when applied in the presence of ATP. In the absence of ATP, genistein, like the other inhibitors, had no effect on cGMP sensitivity, whether it was applied at 10 (Fig. 1 B) or 1 min (n = 12; data not shown) after patch excision. Presumably, when ATP is absent, the PTK is not enzymatically active, precluding the inhibitors from affecting the phosphorylation state, and hence the cGMP sensitivity of the channels.
|
In contrast to their effects on cGMP sensitivity, lavendustin A and erbstatin have no effect on the current elicited by saturating cGMP. Thus, both in the presence (Fig. 1 C) and absence (Fig. 1 D) of ATP, these agents had no effect on the maximal current. However, genistein does have an effect. Addition of 10 µM genistein inhibits the maximal current by ~50% in the presence and by 85% in the absence of ATP. Thus genistein more effectively inhibits maximal current in the absence than in the presence of ATP. These observations indicate that genistein, distinct from the other inhibitors, must do more than simply prevent tyrosine phosphorylation.
Genistein Preferentially Inhibits Closed CNG Channels
We more closely examined the effects of genistein on the maximal current of rod CNG channels expressed in Xenopus oocytes. Excised inside-out patches typically contained hundreds of CNG channels that could be saturated by application of 2 mM cGMP, generating large currents that reach steady state within 100 ms (Fig. 2 A). In the presence of 100 µM genistein, the steady state current elicited by cGMP was much smaller (40% of control) and activated over a more complex time course (Fig. 2 B). A fraction of the total current (4.5%), labeled "residual current," activates over the normal rapid time course. The remaining current activates very slowly, with a time course that could be fit with a single exponential with a time constant of 10.6 s at 100 µM genistein. The observation that pretreatment with genistein slows activation elicited by cGMP suggests that genistein can interact with the CNG channel in its closed state.
|
Genistein can also inhibit the rod CNG channel after
it has been activated by cGMP (Fig. 2 C). In fact, the
steady state level of inhibition is the same regardless of
the order of application of genistein and cGMP.
Genistein slowly suppresses the saturating current with
a time course that can be fit with a single exponential with a time constant of 7.3 s. The inhibition of closed
CNG channels also develops slowly (data not shown),
and the time course can be fit with a single exponential
of 8.9 s. The slow time constant of genistein inhibition
suggests that rather than interacting directly with the
channel, genistein may bind to a distinct protein that
can allosterically influence channel gating. Daidzein,
an inactive analogue of genistein (Akiyama et al.,
1987), has no effect on rod CNG channels (Fig. 2 D).
Moreover, we observed no effect of genistein or daidzein on rat olfactory CNG channels (Dhallan et al.,
1990
) expressed in Xenopus oocytes (n = 10 patches).
To investigate the interaction between genistein and closed CNG channels, patches were pretreated with various concentrations of genistein before application of cGMP, as illustrated in Fig. 2 B. We found that the magnitude of the residual current is inversely related to the genistein concentration (Fig. 3 A). Therefore, the residual current appears to reflect the fraction of the current not blocked by genistein. At saturating concentrations of genistein (>100 µM), the residual current was nearly eliminated, indicating complete inhibition of closed channels by genistein.
|
In contrast to its effect on closed channels, genistein was less effective at inhibiting CNG current once it had been activated by cGMP (Fig. 3 B). Even at high concentrations of genistein (e.g., 1 mM), inhibition of steady state CNG current was incomplete. A comparison of the effect of genistein on the nonactivated CNG channels and the fully activated CNG current for this experiment is shown in Fig. 3 C. Inhibition of the closed channel by genistein was complete and had an apparent Ki of 4.1 µM, which was determined by measuring the residual current. Inhibition of the fully activated current by genistein was incomplete, with a Ki of 72.5 µM, which was determined by measuring the steady state current in the presence of genistein. These results suggest that genistein has a much higher affinity for the closed than for the open CNG channel.
Activation of the rod CNG channel by cGMP involves distinct ligand binding and channel gating steps. Like cGMP, cAMP binds to the rod channel, but it is much less effective at causing channel opening. To distinguish whether the reduced apparent affinity of genistein in the presence of cGMP is due to bound ligand per se, or to the conformational changes associated with channel opening, we investigated the effect of genistein on channels exposed to saturating cAMP. In these experiments, genistein was applied to the patch in conjunction with 20 mM cAMP, which should have saturated the cyclic nucleotide binding sites. Then, the cAMP was rapidly replaced with cGMP to measure the residual current. Our results showed that saturating cAMP had only a small effect on the apparent affinity of genistein, much less than that observed with saturating cGMP (Fig. 3 C).
We also examined the effect of genistein on native
CNG channels from rod outer segments from salamander retina. As we observed for the expressed channel, application of 100 µM genistein on inside-out
patches containing native channels reduced the magnitude of the cGMP-activated current and slowed the activation kinetics (Fig. 3 D). Thus, even though the native
channel contains and
subunits (Kaupp et al., 1989
;
Chen et al., 1993
; Körschen et al., 1995
), it is inhibited
by genistein similarly to the inhibition of the expressed
channel, which contains only
subunits.
Our results suggest that genistein is not a competitive inhibitor of the cyclic nucleotide binding site on the rod channel. The observation that saturating concentrations of genistein incompletely inhibit the steady state CNG current is consistent with this conclusion. Moreover, the extent of inhibition does not decrease as cGMP concentration is increased beyond the level required to saturate the channel. Hence, inhibition is the same in the presence of 2 and 20 mM cGMP, both for low (10 µM) and high (100 µM) concentrations of genistein (Fig. 4 A). Thus, rather than interfering with cyclic nucleotide binding, genistein appears to inhibit the rod CNG channel by allosterically affecting channel gating. Unlike tetracaine, which elicits a voltage-dependent inhibition of CNG channels by binding within the pore of the channel, genistein inhibits the rod channel in a voltage-independent manner (Fig. 4 B).
|
Adenosine Triphosphates Decrease Genistein Inhibition
The inhibition of CNG current by genistein described above occurs in the absence of ATP and therefore cannot involve a phosphorylation reaction. However, we noticed that ATP reduces the ability of genistein to inhibit the channels. Fig. 5 A shows that ATP shifts the dose-response curve for genistein inhibition to the right, such that a higher concentration of genistein is required to inhibit CNG current activation by cGMP. Analysis of these curves (Fig. 5 B) shows that in the absence of ATP, the Ki for genistein inhibition of CNG current activation by cGMP was 4.1 ± 0.7 µM, whereas in the presence of 1 mM ATP, the Ki was shifted to a higher value (10.6 ± 1.3 µM). The Hill coefficient for genistein inhibition was the same in the presence and absence of ATP (2.0 ± 0.2 and 1.9 ± 0.2, respectively). To determine whether the ability of ATP to alter the Ki for genistein involves a phosphorylation reaction, we used a nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMP-PMP), which cannot be used as a substrate in phosphorylation reactions, but which does bind to ATP binding sites in proteins. Like ATP, AMP-PNP reduced the ability of genistein to inhibit the CNG current. Addition of AMP-PNP shifted the Ki to 9.3 ± 0.9 µM without altering the Hill coefficient.
|
To further test whether the phosphorylation state of
the CNG channel is important for determining the Ki
for genistein, we examined CNG channels at various
times after patch excision from the oocyte. During first
10 min after patch excision, the sensitivity of rod CNG
channels to cGMP changes dramatically, resulting from tyrosine dephosphorylation (Molokanova et al., 1997),
probably of the CNG channel protein itself (Molokanova, Maddox, Luetje, and Kramer, manuscript submitted for publication). Hence, at 1 min after excision,
a patch is likely to have a greater proportion of phosphorylated channels than at 10 min after excision. Fig. 6 shows that the Ki values for genistein at 1 and 10 min
after excision do not differ, suggesting that sensitivity
of the channels to genistein is not strongly dependent
on phosphorylation.
|
Since it appears that these effects of ATP on genistein inhibition do not involve changes in phosphorylation, an alternate possibility is that ATP competes with genistein for binding to a common site. We propose that the competition between ATP and genistein concerns the ATP binding site on the PTK. According to this model, genistein, rather than having a direct effect on the rod CNG channel, indirectly regulates gating of the channel by interacting with this tightly associated protein.
Erbstatin Decreases Genistein Inhibition
To further test whether genistein inhibition of the rod
CNG channel involves an interaction with a PTK, we
used the specific PTK inhibitor erbstatin. Studies have
shown that erbstatin competes with peptide substrates
of PTKs, suggesting that erbstatin binds to a site on the
enzyme that normally binds to target proteins (Umezawa
et al., 1986; Imoto et al., 1987
). If the inhibition of the
rod channel by genistein is an indirect effect mediated by a PTK, then erbstatin, like ATP, should reduce the
effect of genistein.
Fig. 7 A shows the effect of erbstatin on genistein inhibition of closed rod CNG channels. In this experiment, genistein was applied on closed CNG channels in the presence or absence of erbstatin, and the channels were subsequently activated by cGMP. Without erbstatin present, 100 µM genistein inhibited closed channels by >95%, resulting in a residual current that was <5% the magnitude of the maximal CNG current. With 100 µM erbstatin present, genistein inhibited closed channels by ~80%, with a residual current 20% of maximal. Group data showing inhibition caused by different concentrations of genistein with and without erbstatin is shown in Fig. 7 B. Below saturation, increasing the concentration of genistein from 10 to 100 µm resulted in increasing inhibition, which was partly suppressed by erbstatin. However, unlike the effect of ATP, the inhibitory effect of erbstatin could not be overcome by adding additional supersaturating concentrations of genistein. Thus, increasing the concentration of genistein 10-fold further from 100 to 1,000 µM did not elicit more inhibition in the presence of erbstatin, indicating that the interaction between erbstatin and genistein is noncompetitive.
|
The ability of erbstatin to depress the action of genistein is more dramatic when the CNG channels are fully activated by cGMP (Fig. 8 A). Genistein inhibition of fully activated CNG current was reduced from 45.8 ± 5.7 to 5.4 ± 1.7% by pretreatment with 100 µM erbstatin (Fig. 8 B). The suppression of genistein inhibition by erbstatin reversed fully after erbstatin was removed from the superfusion solution.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rod CNG channels expressed in Xenopus oocytes appear to be associated with PTKs and PTPs that are constitutively active in excised patches (Molokanova et al.,
1997). Phosphorylation and dephosphorylation catalyzed by these enzymes alter the apparent affinity of the
channels for cGMP. Recent studies strongly suggest that the
subunit channel protein itself is the substrate
for phosphorylation, with a specific tyrosine residue
(Y498) in the cytoplasmic carboxyl domain being a particularly important phosphorylation site (Molokanova,
Maddox, Luetje, and Kramer, manuscript submitted for publication). Ion channels often exist in tight association with protein kinases and phosphatases, such that
complexes of these proteins remain intact even after reconstitution into artificial lipid bilayers (Reinhart and
Levitan, 1995
). A variety of channels have been found
to be biochemically and functionally associated with
PTKs and PTPs (Prevarskaya et al., 1995
; Holmes et al.,
1996
; Wang and Salter, 1994
; Yu et al., 1997
). These accessory proteins can modulate ion channel activity, both
by catalyzing changes in phosphorylation state (Wang
and Salter, 1994
; Aniksztejn et al., 1997
) and by allosterically regulating channel gating (Holmes et al., 1996
).
Our results suggest that genistein affects rod photoreceptor CNG channels expressed in Xenopus oocytes in two ways. First, like other PTK inhibitors, genistein prevents tyrosine phosphorylation of rod CNG channels, allowing the channels to become dephosphorylated and more sensitive to cGMP. Second, unlike other PTK inhibitors, genistein slows activation and reduces the current elicited by saturating cGMP. This second action of genistein does not require ATP, and thus cannot involve inhibition of phosphorylation by PTKs.
The observation that genistein slows activation and
reduces the maximal cGMP-elicited current suggests
that genistein stabilizes the CNG channel in a closed
state. At first glance, genistein appears to inhibit the
rod CNG channel through a mechanism similar to that
employed by the local anesthetic tetracaine (Fodor et al.,
1997a). Tetracaine preferentially binds to the closed
channel with an apparent affinity of ~10 µM. However,
tetracaine block is rapid (<100 ms) and voltage-dependent. Tetracaine is positively charged and mutagenesis
studies suggest that block involves a direct interaction
with a specific glutamate residue in the pore of the rod
channel (Fodor et al., 1997b
). In contrast, genistein inhibition is slow and voltage independent. Moreover,
our evidence suggests that rather than binding directly
to the channel, the genistein inhibition is indirect, involving an accessory protein that allosterically affects
the CNG channel.
The evidence that genistein inhibits the rod channel
by binding to an accessory protein is indirect, but nonetheless compelling. First, the effect of genistein is inhibited in a competitive manner by ATP and AMP-PNP.
There is no evidence that CNG channels possess ATP
binding sites, and ATP itself has no noticeable effect on CNG channels, other than being a substrate in phosphorylation reactions. Thus, if PTK inhibitors are
present, ATP has no effect on the apparent affinity or
maximal current elicited by cGMP (Molokanova et al.,
1997), and does not alter the kinetics of activation or deactivation (our unpublished observations). In contrast, CNG channels expressed in Xenopus oocytes are
closely associated with PTKs and, like all kinases, these
proteins do possess ATP binding sites. Moreover, genistein is known to be a competitive inhibitor of these
binding sites (Akiyama et al., 1987
).
Second, the effect of genistein is decreased in a noncompetitive manner by erbstatin. Again, application of
erbstatin in the absence of ATP fails to reveal any direct
effect on the rod channel. In contrast, erbstatin is a
well characterized inhibitor of PTKs. Studies have
shown that erbstatin is a competitive inhibitor with respect to the substrate binding site on PTKs, whereas it
has properties of both a competitive and a noncompetitive inhibitor with respect to the ATP binding site (Imoto et al., 1987; Posner et al., 1994
). Our observation
that erbstatin reduces genistein inhibition of CNG
channels noncompetitively suggests that these two
agents bind to nonoverlapping sites, consistent with
previous observations about the effects of these inhibitors on PTKs.
Third, the onset of genistein inhibition for both the closed and fully activated rod CNG channel is slower than would be predicted from the apparent affinity and the slow recovery rate. We find that the rod CNG channels require >60 s to recover from genistein inhibition (our unpublished observations). If the on rate of genistein inhibition were diffusion limited, as is the case for many channel blockers and allosteric regulators, an apparent affinity of genistein of 10 µM would suggest that the inhibition rate constant should be <100 ms. The rate of genistein inhibition is much slower than this value, consistent with inhibition resulting from an interaction between the channel and a second protein (a PTK) in the membrane. Despite there being a very large difference in the apparent affinity and magnitude of genistein inhibition of closed versus activated channels, the kinetics of inhibition are similar. This observation is consistent with the hypothesis that the PTK that mediates genistein inhibition diffuses in the membrane, and the on rate of inhibition is determined by the collision rate between this protein and the CNG channel, regardless of the state of the channel.
Finally, genistein inhibition of CNG channels is correlated with the effect of genistein on PTKs. The apparent affinity of genistein for the rod channel (5-80 µM)
is similar to the Ki for inhibition of PTKs (2.6-18 µM;
Akiyama et al., 1987; Umezawa et al., 1990
). The rod
CNG channel can be modulated by PTK and inhibited by genistein, whereas the rat olfactory CNG channel
(Dhallan et al., 1990
) expressed in oocytes exhibits neither effect (our unpublished observations). Closed rod
CNG channels are more susceptible to modulation by
tyrosine phosphorylation (Molokanova, Maddox, Luetje, and Kramer, manuscript submitted for publication)
and are more sensitive to inhibition by genistein, than
are activated channels.
Although many of the physiological effects of genistein, including the inhibition of rod CNG channels
reported here, appear to involve PTKS, there have
been reports of genistein actions that may not involve
PTKs. It has been proposed that genistein directly
blocks voltage-gated Na+ channels in neurons (Paillart
et al., 1997) and smooth muscle (Kusaka and Sperelakis,
1996
), and L-type Ca2+ channels in heart (Chiang et
al., 1996
; Yokoshiki et al., 1996
), in part because the
blocking effect is rapid in onset and reversal and is
mimicked by daidzein, which is an inactive analogue of genistein that does not inhibit PTKs. In contrast, the inhibition of rod CNG channels that we have observed is
slow and is not mimicked by daidzein.
A schematic diagram depicting the action of genistein on closed and open channels is shown in Fig. 9. The closed channel is loosely associated with a PTK, which can phosphorylate specific tyrosine residues in the cyclic nucleotide binding domain (Fig. 9 A), whereas PTK cannot phosphorylate the open channel (Molokanova, Maddox, Luetje, and Kramer, manuscript submitted for publication) (Fig. 9 B). Genistein (G) indirectly affects CNG channels by binding to the PTK, causing a conformational change that stabilizes or alters its interaction with the closed channel (Fig. 9 C). The apparent affinity and efficacy of genistein inhibition are higher for the closed channel than for the open channel (Fig. 9 D). The notion that the PTK more easily dissociates from the open channel is consistent with our findings showing that activation of the channel makes it much less susceptible to tyrosine phosphorylation. Erbstatin binds to the substrate binding site on PTKs, destabilizing the interaction between the PTK and the channel. Hence, in the presence of erbstatin, genistein inhibition is reduced in closed channels (Fig. 9 E) and eliminated in open channels (Fig. 9 F). Despite their ability to block the catalytic activity of PTKs, these effects of genistein are apparently noncatalytic, because they do not require ATP. Moreover, phosphorylated and dephosphorylated channels are equally sensitive to genistein inhibition.
|
In conclusion, our data suggests that when PTKs are
bound to genistein, they can allosterically influence
gating of the rod channel, independently of their role
in catalyzing phosphorylation. Other PTKs have been
shown to noncatalytically regulate gating of ion channels (Holmes et al., 1996; Zeng et al., 1998
). Our finding that genistein inhibits rod CNG channels, not only
in Xenopus oocytes but also in rod outer segments,
raises the possibility that the native rod CNG channel is
associated with native rod PTKs. Indeed, our previous
studies have shown that the native rod CNG channel is
modulated by tyrosine phosphorylation (Molokanova et al., 1997
). It is possible that genistein is not the only
factor capable of triggering noncatalytic inhibition of
rod CNG channels; perhaps there are native ligands
that have a similar effect. Hence, genistein inhibition
of rod CNG channels may have a physiological correlate that is important for photoreceptor function.
![]() |
FOOTNOTES |
---|
Address correspondence to Dr. Richard H. Kramer, P.O. Box 016189, University of Miami School of Medicine, Miami, FL 33101. Fax: 305-243-4555; E-mail: rkramer{at}chroma.med.miami.edu
Original version received 20 July 1998 and accepted version received 9 October 1998.
We thank Dr. Charles W. Luetje for advice and comments on the manuscript.
This work was supported by grants from the National Institutes of Health (EY-11877) and the American Heart Association, Florida Affiliate (9502002) to R.H. Kramer.
![]() |
Abbreviations used in this paper |
---|
CNG, cyclic nucleotide-gated; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Akiyama, T.,
J. Ishida,
S. Nakagawa,
H. Ogawara,
S. Watanabe,
N. Itoh,
M. Shibuya, and
Y. Fukami.
1987.
Genistein, a specific inhibitor of tyrosine-specific protein kinases.
J. Biol. Chem.
262:
5592-5595
|
2. | Aniksztejn, L., S. Catarsi, and P. Drapeau. 1997. Channel modulation by tyrosine phosphorylation in an identified leech neuron. J. Physiol. (Camb.). 498: 135-142 [Abstract]. |
3. | Chen, T.-Y., Y.-W. Peng, R.S. Dhallan, B. Ahamed, R.R. Reed, and K.-W. Yau. 1993. A new subunit of the cyclic nucleotide-gated cation channel in retinal rods. Nature. 362: 764-767 [Medline]. |
4. |
Chen, T.Y.,
M. Illing,
L.L. Molday,
Y.T. Hsu,
K.-W. Yau, and
R.S. Molday.
1994.
Subunit 2 (or beta) of retinal rod cGMP-gated cation channel is a component of the 240-kDa channel-associated
protein and mediates Ca(2+)-calmodulin modulation.
Proc. Natl.
Acad. Sci. USA.
91:
11757-11761
|
5. | Chiang, C.E., S.A. Chen, M.S. Chang, C.I. Lin, and H.N. Luk. 1996. Genistein directly inhibits L-type calcium currents but potentiates cAMP-dependent chloride currents in cardiomyocytes. Biochem. Biophys. Res. Commun 223: 598-603 [Medline]. |
6. | Dhallan, R.S., K.-W. Yau, K.A. Schrader, and R.R. Reed. 1990. Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature. 347: 184-187 [Medline]. |
7. |
Fodor, A.A.,
S.E. Gordon, and
W.N. Zagotta.
1997a.
Mechanism of
tetracaine block of cyclic nucleotide-gated channels.
J. Gen. Physiol.
109:
3-14
|
8. |
Fodor, A.A.,
K.D. Black, and
W.N. Zagotta.
1997b.
Tetracaine reports a conformational change in the pore of cyclic nucleotide-gated channels.
J. Gen. Physiol.
110:
591-600
|
9. | Gordon, S.E., J. Downing-Park, and A.L. Zimmerman. 1995a. Modulation of the cGMP-gated ion channel in frog rods by calmodulin and an endogenous inhibitory factor. J. Physiol. (Camb.). 486: 533-546 [Abstract]. |
10. | Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995b. Diacylglycerol analogs inhibit the rod cGMP-gated channel by a phosphorylation-independent mechanism. Biophys. J. 69: 409-417 [Abstract]. |
11. | Gordon, S.E., and W.N. Zagotta. 1995. A histidine residue associated with the gate of the cyclic nucleotide-activated channels in rod photoreceptors. Neuron. 14: 177-183 [Medline]. |
12. |
Holmes, T.C.,
D.A. Fadool,
R. Ren, and
I.B. Levitan.
1996.
Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.
Science.
274:
2089-2091
|
13. | Hsu, Y.T., and R.S. Molday. 1993. Modulation of the cGMP-gated channel of rod photoreceptor cells by calmodulin. Nature. 361: 76-79 [Medline]. |
14. |
Huang, J.,
M. Nasr,
Y. Kim, and
H.R. Matthews.
1992.
Genistein inhibits protein histidine kinase.
J. Biol. Chem.
267:
15511-15515
|
15. | Ildefonse, M., and N. Bennett. 1991. Single-channel study of the cGMP-dependent conductance of retinal rods from incorporation of native vesicles into planar lipid bilayers. J. Membr. Biol. 123: 133-147 [Medline]. |
16. | Imoto, M., K. Umezawa, K. Isshiki, S. Kunimoto, T. Sawa, T. Takeuchi, and H. Umezawa. 1987. Kinetic studies of tyrosine kinase inhibition by erbstatin. J. Antibiot. (Tokyo) 40: 1471-1473 [Medline]. |
17. | Karpen, J.W., R.L. Brown, L. Stryer, and D.A. Baylor. 1993. Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods. J. Gen. Physiol. 101: 1-25 [Abstract]. |
18. | Kaupp, B.U., T. Niidome, T. Tanabe, S. Terada, W. Bonigk, W. Stühmer, N.J. Cook, K. Kangawa, H. Matsuo, T. Hirode, et al . 1989. Primary structure and functional expression from complementary DNA of the rod photoreceptor cGMP-gated channel. Nature. 342: 762-766 [Medline]. |
19. | Körschen, H.G., M. Illing, R. Seifert, F. Sesti, A. Williams, S. Gotzes, C. Colville, F. Muller, A. Dose, M. Godde, et al . 1995. A 240 kDa protein represents the complete beta subunit of the cyclic nucleotide-gated channel from rod photoreceptor. Neuron. 15: 627-636 [Medline]. |
20. | Kusaka, M., and N. Sperelakis. 1996. Genistein inhibition of fast Na+ current in uterine leiomyosarcoma cells is independent of tyrosine kinase inhibition. Biochim. Biophys. Acta. 1278: 1-4 [Medline]. |
21. | Markovits, J., C. Linassier, P. Fosse, J. Couprie, J. Pierre, A. Jacquemin-Sablon, J.M. Saucier, J.B. Le Pecq, and A.K. Larsen. 1989. Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res. 49: 5111-5117 [Abstract]. |
22. |
Molokanova, E.,
B. Trivedi,
A. Savchenko, and
R.H. Kramer.
1997.
Modulation of rod photoreceptor cyclic nucleotide-gated channels by tyrosine phosphorylation.
J. Neurosci.
17:
9068-9076
|
23. |
Paillart, C.,
E. Carlier,
D. Guedin,
B. Dargent, and
F. Couraud.
1997.
Direct block of voltage-sensitive sodium channels by
genistein, a tyrosine kinase inhibitor.
J. Pharmacol. Exp. Ther.
280:
521-526
|
24. | Posner, I., M. Engel, A. Gazit, and A. Levitzki. 1994. Kinetics of inhibition by tyrphostins of the tyrosine kinase activity of the epidermal growth factor receptor and analysis by a new computer program. Mol. Pharmacol 45: 673-683 [Abstract]. |
25. |
Prevarskaya, N.B.,
R.N. Skryma,
P. Vacher,
N. Daniel,
J. Djiane, and
B. Dufy.
1995.
Role of tyrosine phosphorylation in potassium
channel activation. Functional association with prolactin receptor and JAK2 tyrosine kinase.
J. Biol. Chem.
270:
24292-24299
|
26. | Reinhart, P.H., and I.B. Levitan. 1995. Kinase and phosphatase activities intimately associated with a reconstituted calcium-dependent potassium channel. J. Neurosci. 15: 4572-4579 [Abstract]. |
27. | Ullrich, A., and J. Schlessinger. 1990. Signal transduction by receptor with tyrosine kinase activity. Cell. 61: 203-212 [Medline]. |
28. | Umezawa, H., M. Imoto, T. Sawa, K. Isshiki, N. Matsuda, T. Uchida, H. Iinuma, M. Hamada, and T. Takeuchi. 1986. Studies on a new epidermal growth factor-receptor kinase inhibitor, erbstatin, produced by MH435-hF3. J. Antibiot. (Tokyo). 39: 170-173 [Medline]. |
29. | Umezawa, K., T. Hori, H. Tajima, M. Imoto, K. Isshiki, and T. Takeuchi. 1990. Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors. FEBS Lett. 260: 198-200 [Medline]. |
30. | Wang, Y.T., and M.W. Salter. 1994. Regulation of NMDA receptors by tyrosine kinases and phosphatases. Nature 369: 233-235 [Medline]. |
31. |
Wang, F.,
S. Zeltwanger,
I.C.-H. Yang,
A.N. Nairin, and
T.-C. Hwang.
1998.
Actions of genistein on cystic fibrosis transmembrane conductance regulator channel gating.
J. Gen. Physiol.
111:
477-490
|
32. | Weinreich, F., P.G. Wood, J.R. Riordan, and G. Nagel. 1997. Direct action of genistein on CFTR. Pflügers Arch. 434: 484-491 [Medline]. |
33. |
Yu, X.M.,
R. Ascalan,
G.J. Keil, and
M.W. Salter.
1997.
NMDA channel regulation by channel-associated protein tyrosine kinase Src.
Science.
275:
674-678
|
34. | Yokoshiki, H., K. Sumii, and N. Sperelakis. 1996. Inhibition of L-type calcium current in rat ventricular cells by the tyrosine kinase inhibitor, genistein and its inactive analog, daidzein. J. Mol. Cell. Cardiol. 28: 807-814 [Medline]. |
35. | Zeng, F., M.B. Gingrich, S.F. Traynelis, and P.J. Conn. 1998. Tyrosine kinase potentiates NMDA receptor currents by reducing ionic zinc inhibition. Nat. Neurosci. 1: 185-191 . [Medline] |