Interactions of cyclic nucleotide-gated channel subunits and protein tyrosine kinase probed with genistein.
The cGMP sensitivity of cyclic nucleotide-gated (CNG) channels can be modulated by changes in phosphorylation catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases. Previously, we used genistein, a PTK inhibitor, to probe the interaction between PTKs and homomeric channels comprised of alpha subunits (RETalpha) of rod photoreceptor CNG channels expressed in Xenopus oocytes. We showed that in addition to inhibiting phosphorylation, genistein triggers a noncatalytic interaction between PTKs and homomeric RETalpha channels that allosterically inhibits channel gating. Here, we show that native CNG channels from rods, cones, and olfactory receptor neurons also exhibit noncatalytic inhibition induced by genistein, suggesting that in each of these sensory cells, CNG channels are part of a regulatory complex that contains PTKs. Native CNG channels are heteromers, containing beta as well as alpha subunits. To determine the contributions of alpha and beta subunits to genistein inhibition, we compared the effect of genistein on native, homomeric (RETalpha and OLFalpha), and heteromeric (RETalpha+beta, OLFalpha+beta, and OLFalpha+RETbeta) CNG channels. We found that genistein only inhibits channels that contain either the RETalpha or the OLFbeta subunits. This finding, along with other observations about the maximal effect of genistein and the Hill coefficient of genistein inhibition, suggests that the RETalpha and OLFbeta subunits contain binding sites for the PTK, whereas RETbeta and OLFalpha subunits do not.
Molokanova
E
E
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, FL 33101, USA.
Savchenko
A
A
Kramer
R H
RH
D000557:Ambystoma; D000818:Animals; D006152:Cyclic GMP / Q000494:pharmacology*; D054815:Cyclic Nucleotide-Gated Cation Channels; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D019833:Genistein / Q000494:pharmacology*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018034:Olfactory Receptor Neurons / Q000737:chemistry / Q000201:enzymology; D009865:Oocytes / Q000502:physiology; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry / Q000201:enzymology; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry / Q000201:enzymology; D014981:Xenopus
D054815:Cyclic Nucleotide-Gated Cation Channels; D004791:Enzyme Inhibitors; D007473:Ion Channels; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D006152:Cyclic GMP