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Noncatalytic inhibition of cyclic nucleotide-gated channels by tyrosine kinase induced by genistein.
Molokanova E
,
Savchenko A
,
Kramer RH
.
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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.
Figure 2. Genistein slows activation and reduces maximal current through rod CNG channels. (A) Response of rod CNG channels in an excised patch to saturating (2 mM) cGMP. (B) Response to saturating cGMP after preexposure to 100 μM genistein for 1 min. Inset shows residual current not inhibited by genistein. (C) Partial inhibition of maximal CNG current activated by saturating cGMP. (D) The inactive genistein analogue daidzein has no effect on the maximal current.
Figure 3. Closed channels are more sensitive to genistein inhibition than are activated channels. (A) Inhibition of closed channels by preexposure to various concentrations of genistein for 1 min. Closed channels were activated by application of 2 mM 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 A, B, and D, the letter refers to the genistein concentration key. Note that the amplitude of the residual current is inversely proportional to the genistein concentration. (B) Inhibition of steady state CNG current, maximally activated by saturating cGMP (2 mM). The effects of five different genistein concentrations are indicated by letter to the right of each trace. (C) Doseâinhibition curves of the effect of genistein on closed (residual current as in A) and maximally activated channels (steady state current, as in B). Continuous curves show fits of the data to the Hill equation (see materials and methods). The asterisk depicts inhibition of current activated by saturating cGMP, after preexposure to genistein (10 μm) in conjunction with a saturating concentration of cAMP (20 mM). Note that preexposure to cAMP has only a minor effect on genistein inhibition (n = 6 experiments). (D) Inhibition of native CNG channels from salamander rod outer segments after preexposure to various concentrations of genistein for 1 min. The effects of four different genistein concentrations are indicated by letter to the right of each trace.
Figure 4. Genistein inhibition is not competitive with respect to cGMP and is not voltage dependent. (A) Inhibition of steady state CNG current by 10 and 100 μM genistein. Note that the percent inhibition of the maximal current does not change when cGMP is increased from 2 to 20 mM. (B) Currentâvoltage curves of CNG current activated by saturating cGMP (2 and 20 mM) in the presence and absence of 100 μM genistein. n = 5â8 patches for each experiment.
Figure 5. Competitive effects of ATP and AMP-PNP on genistein inhibition. (A) Doseâ inhibition curves for genistein alone (n = 13), genistein plus 200 μM ATP (n = 7), and genistein plus 200 μM AMP-PNP. Continuous curves show fits to the Hill equation. (B) Histogram showing Ki and Hill coefficient values for genistein inhibition curves. Data represents mean ± SEM for all individual experiments included in A. Note that ATP and AMP-PNP increase the Ki for genistein without affecting the Hill coefficient.
Figure 6. Effect of genistein on the rod CNG channels at different times after patch excision. Genistein was applied on closed rod CNG channels at different times after excision from the oocyte, and residual current was measured after application of cGMP. Bar graph represents mean ± SEM for five patches at 1 and 10 min after excision. During the first 10 min after excision, the K1/2 for cGMP activation gradually declines (see inset), resulting from tyrosine dephosphorylation (Molokanova et al., 1997). The bar graph shows that channels are equally sensitive to genistein, independent of phosphorylation state.
Figure 7. Effect of erbstatin on genistein inhibition of closed channels. (A) CNG current activated by saturating (2 mM) cGMP alone (control) or after preexposure to 100 (a and c) or 1,000 (b and d) μM genistein. Preexposure to 100 μM erbstatin, as indicated, partially inhibits the effect of genistein. (B) Summary data for genistein inhibition alone (open bars) and with erbstatin (filled bars). n = 5 for each condition.
Figure 8. Effect of erbstatin on genistein inhibition of maximally activated channels. (A) Genistein (100 μM) inhibition of CNG current activated by 2 mM cGMP in the absence and presence of 100 μM erbstatin. (B) Summary data for genistein inhibition alone (open bars) and with erbstatin (closed bars). n = 5â8 for each condition.
Figure 9. Schematic diagram of genistein inhibition. (A) The closed channel is loosely associated with a PTK that can catalyze phosphorylation, whereas the open channel (B) is not. Genistein stabilizes the interaction between the PTK and the closed channel, causing inhibition (C), whereas PTK remains unbound from the open channel (D). Erbstatin destabilizes the interaction between the PTK and the channel, decreasing genistein inhibition of the closed channel (E) and eliminating genistein inhibition of the open channel (F). Genistein is depicted as a pentagram labeled G, erbstatin is depicted as an oval labeled E, P represents tyrosine phosphorylation sites on the channel, and the black squares represent ligand (cGMP).
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