Mechanism of inhibition of cyclic nucleotide-gated channel by protein tyrosine kinase probed with genistein

Rod cyclic nucleotide-gated (CNG) channels are modulated by changes in tyro sine phosphorylation catalyzed by protein tyrosine kinases (PTKs) and phosp hatases (PTPs). We used genistein, a PTK inhibitor, to probe the interactio n between the channel and PTKs. Previously we found that in addition to inh ibiting tyrosine phosphorylation of the rod CNG channel alpha -subunit (RET alpha), genistein triggers a noncatalytic inhibitory interaction between t he PTK and the channel. These studies suggest that PTKs affects RET alpha c hannels in two ways: (1) by catalyzing phosphorylation of the channel prote in, and (2) by allosterically regulating channel activation. Here, we study the mechanism of noncatalytic inhibition. We find that noncatalytic inhibi tion follows the same activity dependence pattern as catalytic modulation ( phosphorylation): the efficacy and apparent affinity of genistein inhibitio n are much higher for closed than for fully activated channels. Association rates with the genistein-PTK complex were similar for closed and fully act ivated channels and independent of genistein concentration. Dissociation ra tes were 100 times slower for closed channels, which is consistent with a m uch higher affinity for genistein-PTK. Genistein-PTK affects channel gating , but not single channel conductance or the number of active channels. By a nalyzing single channel gating during genistein-PTK dissociation, we determ ined the maximal open probability for normal and genistein-PTK-bound channe ls. genistein-PTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni2+, whi ch potentiates RET alpha channel gating, partially relieves genistein inhib ition, possibly by disrupting the association between the genistein-PTK and the channel. Studies on chimeric channels containing portions of RET alpha , which exhibits genistein inhibition, and the rat olfactory CNG channel or -subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the geni stein-PTK binding site. Thus, genistein-PTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.