![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rod cyclic nucleotidegated (CNG) channels are modulated by changes in tyrosine phosphorylation catalyzed by protein tyrosine kinases (PTKs) and phosphatases (PTPs). We used genistein, a PTK inhibitor, to probe the interaction between the channel and PTKs. Previously, we found that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel -subunit (RET
), genistein triggers a noncatalytic inhibitory interaction between the PTK and the channel. These studies suggest that PTKs affects RET
channels in two ways: (1) by catalyzing phosphorylation of the channel protein, and (2) by allosterically regulating channel activation. Here, we study the mechanism of noncatalytic inhibition. We find that noncatalytic inhibition follows the same activity dependence pattern as catalytic modulation (phosphorylation): the efficacy and apparent affinity of genistein inhibition are much higher for closed than for fully activated channels. Association rates with the genisteinPTK complex were similar for closed and fully activated channels and independent of genistein concentration. Dissociation rates were 100 times slower for closed channels, which is consistent with a much higher affinity for genisteinPTK. GenisteinPTK affects channel gating, but not single channel conductance or the number of active channels. By analyzing single channel gating during genisteinPTK dissociation, we determined the maximal open probability for normal and genisteinPTK-bound channels. genisteinPTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni2+, which potentiates RET
channel gating, partially relieves genistein inhibition, possibly by disrupting the association between the genisteinPTK and the channel. Studies on chimeric channels containing portions of RET
, which exhibits genistein inhibition, and the rat olfactory CNG channel
-subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the genisteinPTK binding site. Thus, genisteinPTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.
Key Words: cyclic GMP, protein kinase, ion channel gating, kinetics, rod photoreceptor
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyclic nucleotidegated (CNG)1 channels are crucial for generating electrical signals during phototransduction in vertebrate photoreceptors. Light triggers a decrease in the cytoplasmic concentration of cGMP, causing CNG channels in the plasma membrane to close. CNG channels are not invariant reporters of the changes in cGMP concentration, but rather the sensitivity of CNG channels to cGMP can be modulated by intracellular and extracellular signals. Intracellular Ca2+ causes a decrease in cGMP sensitivity of rod CNG channels, mediated by direct binding of calmodulin and other Ca2+-binding proteins to the CNG channel (-subunits of the rod CNG channel (
Remarkably, it appears that PTKs can affect rod CNG channels not only by catalyzing phosphorylation, but also through allosteric regulation by a direct proteinprotein interaction. This conclusion came to light while using genistein, a PTK inhibitor. Genistein competes with ATP binding to PTKs, but does not compete with protein substrates that bind to PTKs at a distinct site (
Activation of CNG channels is a result of conformational changes in protein structure in response to ligand binding to the cytoplasmic cyclic nucleotidebinding domains. How does PTK affect channel activation? After exposure to genistein, the PTK might specifically reduce agonist binding affinity by altering the geometry of the cyclic nucleotide binding site. Alternately, the PTK might impose conformational constraints on the channel protein, hindering channel gating. The goal of this study is to distinguish between these possibilities and clarify the mechanism of genisteinPTK inhibition of rod CNG channels.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression and Recording of CNG Currents
CNG channels were expressed in Xenopus laevis oocytes. Oocytes were injected with 50 nl containing either 1 ng/µl RNA (for single-channel experiments) or 50 ng/µl RNA (macroscopic currents) encoding the -subunit of the bovine retinal rod CNG channel (RET
;
-subunit of the rat olfactory CNG channel (OLF
;
). Inside-out membrane patches usually containing 100200 channels were studied in symmetrical control solution containing (in mM): 115 NaCl, 5 EGTA, 1 EDTA, and 5 HEPES, pH 7.5 with NaOH. cGMP and/or genistein were added to the intracellular control solution. EDTA and EGTA were excluded from Ni2+-containing solutions. After formation of a gigaohm seal, inside-out patches were excised and the patch pipet was quickly (<30 s) placed in the outlet of a 1-mm-diam tube for cGMP application. We used a perfusion manifold containing up to eight different solutions that is capable of solution changes within 50 ms. cGMP was obtained from Sigma-Aldrich, and genistein was obtained from LC Laboratories.
Data Acquisition and Analysis
Current responses through CNG channels were obtained with a patch-clamp (model Axopatch 200A; Axon Instruments), digitized, stored, and later analyzed on a Pentium PC using pClamp 6.0 software. Membrane potential was held at -75 mV. Current responses were normalized to the maximal CNG current (Imax), elicited by saturating (2 mM) cGMP. Normalized doseresponse curves were fit to the Hill equation: I/Imax = 1/(1 + (K1/2/A)n), where A is the cGMP concentration and n is the Hill coefficient, using a nonlinear least squares fitting routine (Origin; Microcal Software, Inc.). 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, and 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 is expressed as mean ± SEM.
Single Channels
Single CNG channels in membrane patches from mRNA-injected oocytes incubated at 18°C first appeared 1218 h after mRNA injection. After this low level of expression was reached, the incubation temperature was reduced to 4°C to stop further expression. Single CNG channel currents were recorded from excised inside-out membrane patches using borosilicate glass pipets coated with Sylgard (Sigma-Aldrich) and fire-polished to resistance of 510 M. The experiments were conducted at room temperature (2022°C).
Membrane potential was held at -80 mV. Single channel events were sampled at 25 kHz and low-pass filtered at 5 kHz through an eight-pole Bessel filter. The opening and closing event was idealized by measuring the amplitude and dwell time, using the half-amplitude threshold detection technique (PClamp6; Axon Instruments). All-points amplitude histograms were constructed from at least 40 s of continuous data recordings and fit by the sum of two Gaussian functions, representing the closed and open states, and used to determine the amplitude of single-channel currents.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genistein Inhibition of Closed and Open CNG Channels
CNG channels are normally activated within several milliseconds of application by direct binding of cyclic nucleotides. In excised patches, the limiting factor in activation is diffusion of the ligand to the binding sites. In the presence of genistein, channel activation is slowed dramatically and the steady-state cGMP-activated current is reduced.
To examine the interaction between closed channels and genisteinPTK, various concentration of genistein were preapplied on patches for 1 min followed by application of saturating cGMP (Fig 1 A). At subsaturating genistein concentrations, a fraction of the total current, which we call "residual current," activates over the normal rapid time course. The magnitude of the residual current is inversely proportional to genistein concentration, presumably reflecting the fraction of channels devoid of genistein (
|
Analyzing the interaction between genisteinPTK and open channels is more difficult because, even with saturating cGMP present, the rod CNG channels flicker between open and closed states. As an alternative to studying open channels, we studied fully activated channels, by applying various concentrations of genistein in the presence of saturating cGMP and recording steady-state currents (Fig 1 B). For both closed and fully activated channels, genistein inhibition was proportional to genistein concentration, and the steady-state level of inhibition was the same regardless of the order of genistein and cGMP application.
Amplitudes of the residual currents (effect on closed channels) and steady-state currents (effect on fully activated channels) were used to generate doseinhibition curves for genistein (Fig 1 C). Comparison of closed versus fully activated channels reveals dramatic differences. First, the efficacy of genistein inhibition was higher for closed channels, with inhibition being nearly complete (98 ± 3%, n = 34) versus incomplete for fully activated channels (65 ± 6%, n = 28). Second, genistein had a much higher apparent affinity for closed channels, with Ki values of 4.3 ± 0.9 µM (n = 22) for closed channels and 84.1 ± 6.8 µM (n = 16) for fully activated channels. Thus, genistein inhibition of cGMP-activated currents exhibited activity-dependent pattern demonstrated previously for tyrosine phosphorylation of CNG channels (
We also observed a difference in the Hill coefficient of genistein inhibition of closed and fully activated channels. The Hill coefficient of genistein inhibition was 1.97 ± 0.08 (n = 22) for closed channels and 1.02 ± 0.03 (n = 16) for fully activated channels (Fig 1 D). This observation suggests that closed channels require two genisteinPTK complexes for inhibition, whereas one is sufficient for inhibiting fully activated channels. However, even in the presence of saturating cGMP, a fraction of the channels will be in their closed state because the maximal open probability is <1.0. The resulting mixed population of open and closed channels with different apparent affinities for genistein could result in an underestimate of the actual numbers of genistein molecules required to inhibit open channels (
Kinetics of GenisteinPTK Inhibition
The observed difference in the apparent affinity of genisteinPTK for open versus closed channels must reflect state-dependent differences in association and/or dissociation between the complex and the channel. To characterize the kinetics of genisteinPTK association with closed channels, we recorded a series of residual cGMP-activated currents at various times after genistein pretreatment (Fig 2 A). The genistein association time course was reconstructed by plotting residual current amplitude as a function of pretreatment duration. The association time course for closed channels could be fit with a single exponential function with time constants (a) of 9.3 s. The time course of genistein inhibition of fully activated channels, determined by applying genistein after steady-state activation by saturating cGMP, was fit with a single exponential with
a = 13.1 s (Fig 2 B).
|
The time required for genistein inhibition presumably reflects a multistep process, including the time necessary for genistein to find its target, PTK (which is dependent on genistein concentration) and the time required for PTK to affect CNG channels (which may be independent of genistein concentration). We found that the association time constant, for both closed and fully activated channels, is independent of genistein concentration. For closed channels, the a values for 10, 25, and 100 µM genistein were 9.1 ± 0.4 (n = 3), 8.9 ± 0.6 (n = 3), and 8.6 ± 0.7 s (n = 6), respectively. For fully activated channels, the
a values for 25, 100, and 250 µM genistein were 12.1 ± 0.6 (n = 5), 12.6 ± 0.7 (n = 12), and 12.6 ± 0.9 s (n = 3), respectively. Therefore, our observation suggests that this second step, interaction of genisteinPTK with the channel, is the rate-limiting step of genistein inhibition.
The association rates of genistein are similar for closed and fully activated channels. To account for the dramatically different affinity of genisteinPTK for closed versus open channels, we expect that dissociation rates should exhibit more substantial differences. To test how channel opening affects the genisteinPTK dissociation rate, we used the following procedure. Genistein was preapplied for 1 min in the absence of cGMP, ensuring that all closed channels were genisteinPTK-bound. For analysis of dissociation from closed channels (Fig 2 C), genistein was washed away and the level of remaining genistein inhibition was estimated by very briefly (1 s) applying saturating cGMP at 15-s intervals during 3545 min of recording. For analysis of dissociation from fully activated channels, genistein was immediately replaced with saturating cGMP (Fig 2 D). This procedure revealed a dramatic state-dependent difference in the apparent dissociation rate. For the fully activated channels, changes in the cGMP-activated current could be fit with a single exponential function with a time constant of 14.4 s. However, for closed channels, two exponentials were required with time constants of 66 and 808 s. Furthermore, the dissociation rates for closed channels were probably underestimated, because the estimated dissociation of genistein was speeded up by short cGMP application to make the changes in the inhibition level visible. Despite the fact that these channels were not continuously maintained in a closed state, genisteinPTK dissociation was still profoundly slower than that for fully activated channels. To ensure that CNG channels were still fully functional in these experiments, long applications of saturating cGMP opened the channels and accelerated genisteinPTK dissociation, resulting in full recovery of the cGMP-activated current with time constant of 2.4 s (Fig 2 C).
Channels in the Open State Can Be Inhibited by Genistein
Genistein is much more potent at inhibiting closed than fully activated rod CNG channels. Are open channels affected by genistein, or is genistein inhibition in the presence of cGMP exclusively due to inhibition during brief closures that occur even with saturating cGMP? With saturating cGMP, rod CNG channels have a maximal open probability of 0.9, indicating that 10% of the channels are closed at any given moment. Since this fraction is subject to strong and very slowly reversible genistein inhibition, the number of inhibited closed channels would accumulate over time until association and dissociation of genisteinPTK from the channels reached equilibrium. Therefore, the apparent inhibition of open channels might be entirely a result of brief closures even in the presence of saturating cGMP.
To address whether channels in their open state can be inhibited by genisteinPTK, we simulated genistein inhibition (Fig 3 A, dotted lines) using the dissociation and association rates determined in Fig 2. Simulations were run assuming that only closed channels are subject to genistein inhibition, or that genistein inhibits open and closed channels equally. A comparison of the actual current recording in Fig 3 A with the predicted current (assuming exclusive inhibition of closed channels) shows that genistein inhibition is faster and more complete than predicted by this model, suggesting that inhibition of open channels also contributes. However, the actual speed and extent of inhibition are smaller than the values predicted for equal effects on open and closed channels. Hence, although open channels appear to be susceptible to inhibition by genisteinPTK, the effects appear to be smaller than for closed channels.
|
To reveal the current flowing through open-bound channels, fully activated channels were first inhibited with genistein, and then briefly closed by removing cGMP and genistein for 4 s (Fig 3 B). Since the dissociation rate of genisteinPTK from closed channels is very slow (>1 min; Fig 2 C), very few of the closed channels should recover from genistein inhibition during this period. When the channels were reopened by subsequent application of saturating cGMP, there appeared a rapidly activating residual current, presumably reflecting channels without genisteinPTK bound (open channels). Application of various concentrations of cGMP showed that the residual current exhibits the same doseresponse relationship for activation as do normal channels in the absence of genistein, further supporting the conclusion that the rapidly activating fraction of current is generated by channels devoid of genisteinPTK. Close inspection reveals that the residual current is smaller than the preceding steady-state current before genistein and cGMP had been washed away, indicating that a population of open-bound channels must have contributed to the steady-state current. In fact, at least 40% of the steady-state current flows through open channels that are genisteinPTK-bound, whereas <60% comes from channels devoid of genisteinPTK.
To determine the sensitivity of open-bound channels to cGMP, we performed the experiment illustrated in Fig 3 B with different concentrations of cGMP, and measured the current flowing through open-bound channels as a fraction of the total control current, which was measured before genistein application (Fig 4 A). As compared with control channels, open-bound channels exhibited a fourfold decrease in the K1/2 for cGMP, whereas the Hill coefficients were similar (Fig 4 B). These results suggest that genistein does not affect the apparent stoichiometry or cooperativity of cGMP binding and agree with our previous studies (
|
Effect of GenisteinPTK on Single Channels
To further distinguish between kinetic states, we analyzed the single channel behavior of rod CNG channels with and without genistein present. Current carried by CNG channels can be described with the equation I = i x N x Po, where i is single-channel current, N is a number of active channels in the patch, and Po is the open probability. Our first goal was to determine which of these parameters is affected by genistein. Single-channel currents activated by saturating cGMP in the absence (Fig 5 A) and in the presence (Fig 5 B) of 100 µM genistein appeared to have the same average amplitudes, as confirmed by all-point histograms, indicating that single-channel current was unaffected by genistein. Likewise, in patches containing 23 channels, genistein did not cause an all-or-none dropout of individual channel activity, indicating that genistein does not affect the number of active channels. However, Fig 5 shows that genistein dramatically alters the Po. The control activity pattern exhibited a nearly constant Po of 0.9, but after genistein application, long closures appeared, such that activity exhibited a "bursty" pattern with 1530 s of high Po (0.40.9) interspersed with 1080-s silent periods (Po = 0). In a total of eight patches without genistein present (>10 min of total recording time of single channel activity in saturating cGMP) long closures were never observed, whereas long closures were consistently observed in each of the six patches with genistein present (
20 min total recording time). In these six patches, genistein caused a decrease in the average Po of 39 ± 6%, fully accounting for the 41% inhibition of macroscopic current induced by 100 µM genistein.
|
While the control gating pattern is relatively simple, in the presence of genistein, gating appears to fluctuate from one mode to another, each with distinct gating characteristics. These modes may correspond to genistein-bound and unbound states, but at steady state, we have no way to relate channel activity to specific kinetic states. Therefore, we examined the behavior of genistein-inhibited rod CNG channels immediately after removal of genistein. Naive channels that had not been preexposed to genistein activate rapidly with little delay (<1 s) upon exposure to saturating cGMP, and exhibit uniform gating with high Po. In contrast, channels preexposed to genistein exhibited a prolonged delay in activation upon application of cGMP, followed by bursts of channel openings, and eventually followed by a return to the normal (genistein-free) gating pattern (Fig 6A and Fig B). The transition from fully inhibited to normal gating appeared to involve discrete steps. Fig 6 A shows an example of the activity pattern seen in three out of seven experiments, where the Po shifted in a relatively abrupt manner from a very low value (0.02 ± 0.01) to 0.9 with only a brief period (<3 s) of intermediate Po values. Fig 6 B shows an example of channel behavior seen in the remaining four patches, where the Po was initially near zero and then shifted in two distinct steps. The first appeared as a burst of openings and had an average Po of
0.35, followed by an increase to 0.9. Analysis of all four patches that exhibited an intermediate gating mode indicated that the Po for this mode was 0.39 ± 0.08. The abrupt changes in the average Po were irreversible, such that channel activity increased in discrete steps, but never decreased again, as long as genistein was not reapplied. The irreversible jumps in open probability are consistent with the stepwise dissociation of genistein and/or PTK. The diagrams illustrate the channel configuration that we propose corresponds to each of the gating modes. As suggested previously (
|
Genistein Changes the Free Energy Associated with Channel Opening
To help clarify the mechanism of genistein inhibition, we used a model for channel activation that involves the cooperative binding of cyclic nucleotides, followed by an allosteric opening transition (Gopening) is related to L by the equation
Gopening = -RT ln (1/L). According to this model, genisteinPTK changed
Gopening from -1.65 to 0.24 kcal/mol (one genistein molecule) and 2.21 kcal/mol (two genistein molecules). Hence, genistein inhibits rod CNG channels by dramatically raising the
G for the allosteric opening transition, such that it becomes an energy-requiring rather than an energy-yielding reaction.
Interaction between Genistein and Ni2+ Effects on Rod CNG Channels
To further characterize the changes in the channel behavior elicited by genistein, we tested the effect of 10 µM Ni2+ on the channels whose gating was impaired by genistein. Ni2+ has been shown to promote rod CNG channel opening by binding to a specific histidine on the COOH terminus of the channel protein and stabilizing the open state (
|
We have considered two nonmutually exclusive explanations that might account for how Ni2+ reduces genistein inhibition. First, since Ni2+ dramatically increases the open probability, perhaps the attenuation of genistein inhibition results solely from the fact that genistein affects open channels less strongly than closed channels. However, we have shown that genistein has a finite ability to inhibit open channels. Therefore, the complete absence of genistein inhibition in the presence of Ni2+ suggests that an additional mechanism must be involved.
To determine the extent to which the effect of Ni2+ on genistein inhibition results from the increase in open probability, we calculated the extent of genistein inhibition and plotted it as a function of open probability. Curves relating cGMP concentration to the extent of genistein inhibition (Fig 8 A) were transformed into curves relating open probabilities to the extent of genistein inhibition (Fig 8 B) by assuming that with Ni2+ present, saturating cGMP leads to an open probability very close to 1.0. Hence, data were normalized with respect to the current elicited by 2 mM cGMP plus Ni2+ (Fig 8 B, asterisk). If the channel open probability alone accounts for Ni2+ attenuation of genistein inhibition, then data sets with and without Ni2+ should be indistinguishable. However, at the same values of open probability, genistein inhibition is less for channels activated by cGMP with Ni2+ versus channels activated by cGMP alone. This deviation indicates that an additional effect must be involved in Ni2+ attenuation of genisteinPTK inhibition.
|
Therefore, we suggest that Ni2+ binding alters the physical association between the channel and the PTK, making it more difficult for genisteinPTK to affect channel behavior. We tested this hypothesis using closed channels. Ni2+ associates rather slowly (tens of seconds) with CNG channels, as demonstrated by the slow potentiation shown in Fig 9 A) and by previous studies (
|
Structural Determinants of Genistein Inhibition of Rod CNG Channels
Which part of the rod CNG channel -subunit (RET
) interacts with genisteinPTK and confers inhibition? To address the question, we took advantage of our previous finding that the rat olfactory CNG channel
-subunits (OLF
), which are highly homologous to the rod channel, do not exhibit genistein inhibition (
channel and genisteinPTK, we examined chimeric channels (
|
We first tested the role of specific histidine residues that were previously implicated in Ni2+ effects on CNG channels (. Substitution of H420 in RET
, which is necessary for Ni2+ potentiation, with an asparagine (CHM25) did not reduce genistein inhibition. Thus, CHM25, like its parent RET
channel, showed complete inhibition with genistein applied on closed channels. Likewise, substitution of Q417 with a histidine (CHM35), which is necessary for Ni2+ inhibition of OLF
channels, also did not reduce genistein inhibition. The converse chimeras (CHM34 and CHM 30) in which substitutions were made into the OLF
background also had no effect: neither substitution introduced genistein inhibition.
Exchange of the cyclic nucleotidebinding domains between RET and OLF
channels did not alter genistein inhibition. Thus, RET
channels with the OLF
cyclic nucleotidebinding domain (CHM17) exhibited complete inhibition by genistein, whereas the OLF
channel with a RET
cyclic nucleotidebinding domain (CHM18) exhibited no inhibition. Switching the NH2-terminal domains through part of the first membrane-spanning segment (CHM 15 and CHM16) also had no effect; either chimera behaved like their respective RET
or OLF
parent channel. Substitution from mid-S1 through S4, including all of S2 and S4 also had no effect. Thus, CHM12, which consisted of RET
except for this region was fully inhibited by genistein, whereas CHM 14, which consisted of OLF
except for this region was not inhibited.
We were able to alter genistein inhibition of RET channels by substituting "a pore module" derived from the OLF
channel, including S5, the pore-forming P region, S6, and the initial part of the C-linker flanking S6 (CHM11). In the open state, this chimera was completely insensitive up to 500 µM genistein; in the closed state, maximal inhibition caused by 500 µM genistein was only
15 vs. 98% in wild-type channel, suggesting that this region contains most, but not all, of the sites necessary for genisteinPTK interaction. We tested three parts of the S5-P-S6 region separately. Exchange of S5 domains between RET
and OLF
channels (CHM20 and CHM22) and introduction of the OLF
pore into RET
channel (CHM 19) did not alter genistein inhibition. However, introduction of a region including part of the pore, S6, and part of the C-linker from the RET
into the OLF
channel (CHM23) had a dramatic effect. Indeed, the resulting channel was susceptible to genistein inhibition to the same extent as RET
channels (99% inhibition of closed channels). Thus, introduction of the RET
S6 and flanking regions was the only change capable of conferring genistein inhibition onto the OLF
channel. These results suggest that the region in or around transmembrane domain S6 (namely, amino acids 343421) is crucial for genistein inhibition, and may be the genisteinPTK binding site on the RET
channel.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Noncatalytic Inhibition of CNG Channels by PTK
PTKs can influence RET channels in two ways: (1) by catalyzing phosphorylation, and (2) by noncatalytically inhibiting the channels in the presence of genistein (
All of these observations lead to the proposal that genistein inhibits RET channels by first binding to a PTK, which then impairs channel gating. The observation that the on-rate of genistein inhibition is independent of genistein concentration is consistent with the second step in this process being rate-limiting. It is unclear whether the PTK is stably associated with the channel, or whether the PTK can dissociate and associate with the channel. If the PTK is stably bound to the channel, then the slow kinetics of inhibition must reflect slow conformational changes induced by genistein. If the PTK can dissociate, the slow kinetics might reflect the diffusion of genisteinPTK and binding and unbinding of the complex and the channel.
Genistein is much more effective in inhibiting closed than open channels. While genistein inhibition of closed channels is dramatic and complete, we have shown that open channels can also bind genisteinPTK, causing a less pronounced degree of inhibition. The act of opening, which has been suggested to involve the S6 transmembrane domain, may decrease the stability of binding of channels to genisteinPTK. The association rate of genisteinPTK to either closed and open channels appear to be very similar, whereas dissociation of genisteinPTK occurs much more rapidly from open than from closed channels. Hence, opening channels with cGMP should cause a great acceleration of dissociation, ensuring more rapid recovery from genistein inhibition.
Noncatalytic genistein inhibition occurs not only in RET channels exogenously expressed in oocytes, but also in native CNG channels from rod outer segments (
Structural Basis of Genistein Inhibition
The site in the RET channel that confers genistein inhibition on OLF
channels is a region containing all of the S6 transmembrane domain and neighboring regions of the pore and part of the cytoplasmic C-linker (CHM 23). Genistein inhibition of RET
channels can be greatly reduced, but not completely eliminated, by substitution of a similar and overlapping region spanning from S4 to S6 from OLF
(CHM 11). No other parts of the channel appear crucial for allowing inhibition of RET
or conferring inhibition to OLF
channels. The simplest explanation for these mapping results is that the region in and around S6 is important, but other parts of the RET
channel protein also contribute to the binding site of genisteinPTK. Notably, the tyrosine phosphorylation site (Y498) responsible for catalytic modulation (i.e., phosphorylation) by PTKs (
channel at Y498. However, the binding site for the PTK must involve a much larger protein interface, apparently including a more distant region that may be primarily intramembranous (S6 and flanking regions).
Both genistein inhibition and the ability to modulate rod CNG channels by tyrosine phosphorylation are remarkably stable in membrane patches excised from Xenopus oocytes, persisting unabated for >30 min, even with continuous perfusion. Loosely associated membrane constituents, such as G-protein subunits, cytoskeletal components, and soluble kinases and phosphatases (
Interaction of Ni2+ Potentiation and Genistein Inhibition
Ni2+ increases current through RET channels and increases the apparent affinity for cyclic nucleotides (
How Does the Association between Channel and PTK Affect Channel Behavior?
Allosteric models involving concerted conformational changes between closed and open states, based on the classic model devised for hemoglobin (
Application of genistein on fully activated channels results in a fourfold decrease in cGMP sensitivity. This could result from a decrease in the binding affinity for cyclic nucleotides or from a change in the energetics of channel opening. If genisteinPTK lowered the binding affinity for cGMP, one would expect that, in the presence of genistein, the gating pattern of single channels at high concentrations of cGMP would simply resemble the gating at low concentrations of cGMP. However, genisteinPTK induces a novel gating pattern, consisting of bursts of high open probability interspersed with silent periods, unlike the uniformly low open probability normally seen at low concentrations of cGMP. Hence, an effect on cGMP binding affinity alone cannot account for genistein inhibition. Therefore, at least some of genistein inhibition must result from a change in the process of channel gating.
To quantify the effect of genistein on channel gating, we measured the open probability of single genisteinPTK-bound channels by taking advantage of the slow dissociation rate of the complex from closed channels. At steady state, it is difficult to ascertain the number of genisteinPTK complexes bound to a channel at any given moment. However, by capturing channel gating as genisteinPTK is dissociating, we observed three sequential modes of channel gating, each with progressively higher open probability, as the channel returns to normal activity. At first, the maximal number of genisteinPTK complexes are still bound to the channels. The value of the Hill coefficient for genistein inhibition is 2, and our previous studies ( channels can bind up to two genisteinPTKs. Therefore, we suggest that the initial gating mode, exhibiting the lowest maximal open probability, corresponds to channels with two genisteinPTK complexes. Next, the channels exhibit an intermediate gating mode, corresponding to one genisteinPTK complex bound. Finally, the channel exhibits its normal high open probability, corresponding to complete dissociation of genisteinPTK from the channel. The energetics of channel gating progressively becomes more favorable as the channel sequentially sheds the two genisteinPTK complexes. With two genisteinPTK complexes bound, the
Gopening is positive (+2.13 kcal/mol) and is very close to the value for normal channels with cAMP bound (+2.26 kcal/mol), a very poor partial agonist. Hence, we conclude that by binding to regions of the channel important for gating, genisteinPTK imposes conformational constraints on the channel protein, hindering the ability of the channel to open.
![]() |
Footnotes |
---|
Address correspondence to Dr. Richard H. Kramer, 121 LSA, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720. Fax: (510) 643-6791; E-mail: rhkramer{at}uclink4.berkeley.edu
1 Abbreviations used in this paper: CNG, cyclic nucleotidegated; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Dr. William N. Zagotta for providing chemic channel constructs.
This work was supported by the Young Investigator Award from the National Alliance for Research on Schizophrenia and Affective Disorders to E. Molokanova and by grants from the National Institutes of Health (EY-11877 and EY-12608) to R.H. Kramer.
Submitted: 25 October 2000
Revised: 22 January 2001
Accepted: 22 January 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chen, T.Y., Illing, M., Molday, L.L., Hsu, Y.T., Yau, K.-W., and Molday, R.S. 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
Dhallan, R.S., Yau, K.-W., Schrader, K.A., and Reed, R.R. 1990. Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature. 347:184-187[Medline].
Gordon, S.E., and Zagotta, W.N. 1995a. Localization of regions affecting an allosteric transition in the cyclic nucleotide-activated channels. Neuron. 14:857-864[Medline].
Gordon, S.E., and Zagotta, W.N. 1995b. Subunit interaction in coordination of Ni2+ in cyclic-nucleotide-gated channels. Proc. Natl. Acad. Sci. USA. 92:10222-10226[Abstract].
Gordon, S.E., Brautigan, D.L., and Zimmerman, A.L. 1992. Protein phosphatases modulate the apparent agonist affinity of the light-regulated ion channel in retinal rods. Neuron. 9:739-748[Medline].
Gordon, S.E., Downing-Park, J., and Zimmerman, A.L. 1995a. Modulation of the cGMP-gated ion channel in frog rods by calmodulin and an endogenous inhibitory factor. J. Physiol. 486:533-546[Abstract].
Gordon, S.E., Downing-Park, J., Tam, B., and Zimmerman, A.L. 1995b. Diacylglycerol analogs inhibit the rod cGMP-gated channel by a phosphorylation-independent mechanism. Biophys. J. 69:409-417[Abstract].
Goulding, E.H., Tibbs, G.R., Liu, D., and Siegelbaum, S.A. 1993. Role of H5 domain in determining pore diameter and ion permeation through cyclic nucleotide-gated channels. Nature. 364:61-64[Medline].
Hsu, Y.T., and Molday, R.S. 1993. Modulation of the cGMP-gated channel of rod photoreceptor cells by calmodulin. Nature. 361:76-79[Medline].
Ildefonse, M., and Bennett, N. 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].
Karpen, J.W., Brown, R.L., Stryer, L., and Baylor, D.A. 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].
Kaupp, B.U., Niidome, T., Tanabe, T., Terada, S., Bonigk, W., Stuhmer, W., Cook, N.J., Kangawa, K., Matsuo, H., and Hirode, T. et al. 1989. Primary structure and functional expression from complementary DNA of the rod photoreceptor cGMP-gated channel. Nature. 342:762-766[Medline].
Levitan, I.B. 1999. Modulation of ion channels by protein phosphorylation. Adv. Second Messenger Phosphoprotein Res. 33:3-22[Medline].
Liu, Y., Holmgren, M., Jurman, M.E., and Yellen, G. 1997. Gated access to the pore of a voltage-dependent K+ channel. Neuron. 19:175-184[Medline].
Liu, D.T., Tibbs, G.R., Paoletti, P., and Siegelbaum, S.A. 1998. Constraining ligand-binding site stoichiometry suggests that a cyclic nucleotide-gated channel is composed of two functional dimers. Neuron. 21:235-248[Medline].
Molokanova, E., Trivedi, B., Savchenko, A., and Kramer, R.H. 1997. Modulation of rod photoreceptor cyclic nucleotide-gated channels by tyrosine phosphorylation. J. Neurosci. 17:9068-9076
Molokanova, E., Savchenko, A., and Kramer, R.H. 1999a. Noncatalytic inhibition of cyclic nucleotidegated channels by tyrosine kinase induced by genistein. J. Gen. Physiol 113:45-56
Molokanova, E., Maddox, F., Luetje, C.W., and Kramer, R.H. 1999b. Activity-dependent modulation of rod photoreceptor cyclic nucleotide-gated channels mediated by phosphorylation of a specific tyrosine residue. J. Neurosci. 19:4786-4795
Molokanova, E., Savchenko, A., and Kramer, R.H. 2000. Interactions of cyclic nucleotidegated channel subunits and protein tyrosine kinase probed with genistein. J. Gen. Physiol. 115:685-696
Monod, J., Wyman, J., and Changeux, J.P. 1965. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12:88-118[Medline].
Muller, F., Bonigk, W., Sesti, F., and Frings, S. 1998. Phosphorylation of mammalian olfactory cyclic nucleotide-gated channels increases ligand sensitivity. J. Neurosci 18:164-173
Paoletti, P., Young, E.C., and Siegelbaum, S.A. 1999. C-linker of cyclic nucleotidegated channels controls coupling of ligand binding to channel gating. J. Gen. Physiol. 113:17-34
Ruiz, M., and Karpen, J.W. 1999. Opening mechanism of a cyclic nucleotidegated channel based on analysis of single channels locked in each liganded state. J. Gen. Physiol 113:873-895
Ruiz, M., Brown, R.L., He, Y., Haley, T.L., and Karpen, J.W. 1999. The single-channel dose-response relation is consistently steep for rod cyclic nucleotide-gated channels: implications for the interpretation of macroscopic dose-response relations. Biochemistry. 38:10642-10648[Medline].
Stryer, L. 1987. Visual transduction: design and recurring motifs. Chem. Scr 27B:161-171.
Tibbs, G.R., Goulding, E.H., and Siegelbaum, S.A. 1997. Allosteric activation and tuning of ligand efficacy in cyclic-nucleotide-gated channels. Nature. 386:612-615[Medline].
Womack, K.B., Gordon, S.E., He, F., Wensel, T.G., Lu, C.-C., and Hilgemann, D.W. 2000. Do phosphatidylinositides modulate vertebrate phototransduction? J. Neurosci 20:2792-2799
Zong, X., Zucker, H., Hofmann, F., and Biel, M. 1998. Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channels. EMBO (Eur. Mol. Biol. Organ.) J. 17:353-362