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Mechanism of inhibition of cyclic nucleotide-gated channel by protein tyrosine kinase probed with genistein.
Molokanova E
,
Kramer RH
.
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Rod cyclic nucleotide-gated (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 alpha-subunit (RETalpha), genistein triggers a noncatalytic inhibitory interaction between the PTK and the channel. These studies suggest that PTKs affects RETalpha 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 genistein-PTK 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 genistein-PTK. Genistein-PTK affects channel gating, but not single channel conductance or the number of active channels. By analyzing single channel gating during genistein-PTK dissociation, we determined the maximal open probability for normal and genistein-PTK-bound channels. genistein-PTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni(2+), which potentiates RETalpha channel gating, partially relieves genistein inhibition, possibly by disrupting the association between the genistein-PTK and the channel. Studies on chimeric channels containing portions of RETalpha, which exhibits genistein inhibition, and the rat olfactory CNG channel alpha-subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the genistein-PTK binding site. Thus, genistein-PTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.
Figure 1. Genistein is more potent in inhibiting closed than fully activated RETα channels. (A) Inhibition of closed channels by preexposure to various concentrations of genistein for 1 min. Closed channels were activated by application of saturating cGMP. Residual currents at five different genistein concentrations are indicated by dotted lines, and the genistein concentrations are indicated by letter to the right of each trace. In parts A and B the letter refers to the genistein concentration key. (B) Inhibition of steady-state CNG current, fully activated by saturating cGMP. The five different genistein concentrations are indicated by letter to the right of each trace. (C) Dose–inhibition curves of the effect of genistein on closed (residual current as in part A; n = 36) and fully activated channels (steady-state current, as in part B; n = 28). Continuous curves show fits of the data to the Hill equation. (D) Apparent affinity (Ki) and Hill coefficients derived from the Hill equation fits to dose–inhibition curves for closed and fully activated CNG channels ([open circles] closed channels; [filled circles] fully activated channels, as in Fig. 1 C).
Figure 2. Association/dissociation kinetics of genistein–PTK from closed (A and C) and fully activated (B and D) channels. (A) Association of genistein–PTK with closed channels. Currents activated by saturating cGMP obtained after preexposure to 100 μM genistein for various time intervals (1, 2, 5, 10, 15, 20, 25, 30, and 60 s). Open circles represent the amplitude of residual currents that remain uninhibited by genistein. Dashed curve shows single exponential fit of the data. Inset shows the application protocol. Note that genistein application time was variable. (B) Association of genistein–PTK with fully activated channels. (C) Dissociation of genistein–PTK from closed channels. After 1 min preexposure to genistein, saturating cGMP was puffed onto the patch every 15 s for 1 s (multiple arrows). Open squares indicate residual currents resulting from activation of genistein-free channels (D) Dissociation of genistein–PTK from fully activated channels. After 1 min preexposure to genistein, saturating cGMP alone was applied to record the recovery of activated channels from genistein inhibition.
Figure 3. Open channels participate in genistein inhibition. (A) Fully activated current inhibited by 100 μM genistein superimposed with simulations of models in which (1) only closed channels are inhibited, and (2) closed and open channels are inhibited equally. Note that inhibition of closed channels alone cannot account for observed current. (B) Dissection of open and open-bound states. Channels were first fully activated with saturating cGMP. Since maximal open probability is 0.9, ∼90% of the channels were open and 10% were closed. Genistein was added, cGMP was briefly removed, and then cGMP was reapplied without genistein. Channels that opened rapidly upon reapplication of cGMP (Open channels) were genistein–PTK-free (inset), whereas channels that opened sluggishly were genistein–PTK bound. Since the steady-state current in the presence of genistein was larger than the rapidly activating current upon cGMP reapplication, the difference between these currents represents open channels that were genistein–PTK-bound (Open-Bound).
Figure 4. Genistein alters the apparent affinity of RETα channels for cGMP. (A) Dose–response curves for cGMP activation of CNG channels in the absence (control) and in the presence of 100 μM genistein (Open and Open-Bound channels). Continuous curves show fits to the Hill equation. (B) Apparent affinity (K1/2) for cGMP and Hill coefficients for control (n = 42), genistein-free (Open, n = 22), and genistein-inhibited (Open-Bound, n = 22) channels.
Figure 5. Effects of genistein on single-channel activity. Representative single-channel currents (top), corresponding open probabilities (middle) sampled in 1-s bins, and all-point amplitude histograms of RETα channel activity activated by saturating cGMP (2 mM). Amplitude histograms were fitted by two Gaussians with variances σ2c and σ2o for closed and open distributions, respectively. All data were obtained at −80 mV. (A) Control single-channel currents. Parameters for Gaussians are as follows: σc = 0.19 pA, i =1.96 pA, σo = 0.39 pA, and Po = 0.94. (B) Single-channel current in the presence of genistein. Parameters for Gaussians are as follows: σc = 0.23 pA, i =1.97 pA, σo = 0.38 pA, and Po = 0.529.
Figure 6. Dissociation of genistein–PTK from single channels. (Top) Two examples of single-channel currents after preexposure to 100 μM genistein. Arrows in A and B show the moment (time 0) when superfusion with solution containing 2 mM cGMP genistein was applied, washing away the genistein-containing solution. (Middle) Corresponding plots of open probability. In A, note the very low open probability, followed by an abrupt increase 150 s later, as genistein unbinds. In B, note the two-step increase in open probability, possibly reflecting sequential dissociation of two genistein molecules. Diagrams illustrate tetrameric CNG channels (open symbols), PTK molecules (shaded symbols), and genistein (solid symbols). (Bottom) Amplitude histograms fit with Gaussian distributions. Parameters for Gaussians are as follows: (Α) σc = 0.21 pA, i = 1.97 pA, and σo = 0.36 pA; (B) σc = 0.24 pA, i = 1.96 pA, and σo = 0.38 pA.
Figure 7. Ni2+ attenuates genistein inhibition of RETα channels. (A) Effect of genistein on CNG current fully activated by saturating cGMP, in the absence and presence of 10 μM Ni2+. (B) Effect of genistein on CNG current, partially activated by 25 μM cGMP, in the absence and presence of 10 μM Ni2+. (C) Dose–response curves of channel activation in control, genistein, Ni2+, and genistein plus Ni2+.
Figure 8. Analysis of Ni2+ attenuation of genistein inhibition. (A) Genistein inhibition in the absence and presence of 10 μM Ni2+ at various cGMP concentrations. Genistein inhibition is defined as (control current − current with 100 μM genistein)/control current (n = 6–9 experiments for each point). (B) Genistein inhibition as a function of channel open probability, with and without Ni2+ present. Each data point, taken from the same experiments shown in A, represents mean ± SEM values for percent inhibition of CNG current by genistein, and mean ± SEM of open probability.
Figure 9. Ni2+ binds to closed channels and attenuates genistein inhibition. (A) Ni2+ slowly potentiates channels activated by 25 μM and 2 mM cGMP. (B) Preapplication of Ni2+ on closed channels potentiates subsequent activation by cGMP. (C) Genistein inhibition of closed channels is reduced by preapplication of Ni2+.
Figure 10. Structural determinants of genistein inhibition of RETα channels. (Left) RETα-based channels (shaded) with substituted regions containing OLFα sequences (black portions). (Right) OLFα-based channels (black) with substituted regions containing RETα sequences (shaded portions). Pluses and Minuses represent the presence or absence of genistein inhibition for each channel.
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