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Interactions of cyclic nucleotide-gated channel subunits and protein tyrosine kinase probed with genistein.
Molokanova E
,
Savchenko A
,
Kramer RH
.
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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.
Figure 2. PTK inhibitors reduce the genistein inhibition. (A) Inhibition of closed rod photoreceptor CNG channels by 1 min pre-exposure to 100 μM genistein alone or together with PTK inhibitors (500 μM AMP-PNP or 100 μM erbstatin). Closed channels were activated by application of 2 mM cGMP (control). (B) Inhibition of fully activated channels by genistein alone or together with PTK inhibitors.
Figure 1. Effect of genistein on native rod CNG channels. (A) Activation of CNG channels by saturating cGMP without genistein. (B) Effect of genistein on closed channels. Response to saturating cGMP after pre-exposure to 100 μM genistein for 1 min. The broken line distinguishes the slowly activating current component inhibited by genistein and the rapidly activating residual current. (C) Effect of 100 μM genistein on fully activated channels, indicated by the broken line.
Figure 3. Comparison of genistein inhibition of native CNG channels. Dose-inhibition curves of the effect of genistein on native closed (A) and fully activated (B) CNG channels from rod and cone outer segments and olfactory receptor neurons. Continuous curves in A and B show fits of the data to the Hill equation (see materials and methods). (C) Bar graph showing Ki values for genistein inhibition of closed and activated channels from rods, cones, and olfactory neurons. Data represents mean ± SEM for all of the individual experiments included in A and B.
Figure 4. Subunit dependence of genistein inhibition of closed rod CNG channels. (A) Dose-inhibition curves for genistein effect on closed native rod, homomeric RETα, and heteromeric RETα+β channels. (B) Bar graph showing Ki and Hill coefficient values for genistein inhibition curves. Data represents mean ± SEM for all individual experiments included in A.
Figure 6. Kinetics of inhibition of fully activated homomeric RETα (A) and heteromeric RETα+β (B) channels by various concentrations of genistein.
Figure 5. Subunit dependence of genistein inhibition of fully activated rod CNG channels. (A) Dose-inhibition curves for genistein effect on native and homomeric RETα, and heteromeric RETα+β channels. All channels were fully activated with saturating (2 mM) cGMP before genistein application. (B) Bar graph showing Ki and Hill coefficient values for genistein inhibition curves. Data represents mean ± SEM for all individual experiments included in A.
Figure 9. Schematic diagram of genistein inhibition. Homomeric and heteromeric RET and OLF channels and their interactions with PTKs, symbolized with the shaded crescent symbol. Genistein (G) is depicted bound to the PTK. In the closed state of the homomeric RETα channel (A), two PTKs, and thus two genistein molecules, are required to completely inhibit the channel. cGMP triggers channel opening, which functionally dimerizes RETα subunits. With the PTK spanning two functional dimers, only one PTK, and therefore one genistein, is sufficient to inhibit the channel, although two are still able to bind. In addition, the affinity of the closed channel for the PTK is higher than for the open channel, hence a tighter fit between the channel and the PTK symbol is depicted for the closed channel. For the RETα+β channel (B), only the RETα binds the PTK. Hence there is no change in the number of PTK required to blocked the closed and open channels. The homomeric OLFα channel (C) is unaffected by genistein because OLFα subunits do not contain PTK binding sites. The heteromeric OLFα+OLFβ (D) channel binds one PTK, via the binding sites on the OLFβ subunits.
Figure 7. Subunit dependence of genistein inhibition of olfactory CNG channels. Dose-inhibition curves for genistein effect on closed (A) and fully activated (B) native (n = 3–5), homomeric OLFα (n = 4), and heteromeric OLFα+β (n = 6) channels.
Figure 8. Summary data for genistein inhibition of various expressed homomeric and heteromeric channels in closed (A) and fully activated (B) states. n = 5–8 for each channel type.
Akiyama,
Genistein, a specific inhibitor of tyrosine-specific protein kinases.
1987, Pubmed
Akiyama,
Genistein, a specific inhibitor of tyrosine-specific protein kinases.
1987,
Pubmed
Berghard,
Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs.
1996,
Pubmed
Bielefeldt,
Phosphorylation and dephosphorylation modulate a Ca(2+)-activated K+ channel in rat peptidergic nerve terminals.
1994,
Pubmed
Bradley,
Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP.
1994,
Pubmed
Bönigk,
The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits.
1999,
Pubmed
Chen,
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.
1994,
Pubmed
Chen,
A new subunit of the cyclic nucleotide-gated cation channel in retinal rods.
1993,
Pubmed
,
Xenbase
Dhallan,
Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons.
1990,
Pubmed
Fantl,
Signalling by receptor tyrosine kinases.
1993,
Pubmed
Finn,
Functional co-assembly among subunits of cyclic-nucleotide-activated, nonselective cation channels, and across species from nematode to human.
1998,
Pubmed
Gordon,
Protein phosphatases modulate the apparent agonist affinity of the light-regulated ion channel in retinal rods.
1992,
Pubmed
Grunwald,
Identification of a domain on the beta-subunit of the rod cGMP-gated cation channel that mediates inhibition by calcium-calmodulin.
1998,
Pubmed
Holmes,
Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.
1996,
Pubmed
Imoto,
Kinetic studies of tyrosine kinase inhibition by erbstatin.
1987,
Pubmed
Kaupp,
Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel.
1989,
Pubmed
,
Xenbase
Körschen,
Interaction of glutamic-acid-rich proteins with the cGMP signalling pathway in rod photoreceptors.
1999,
Pubmed
Körschen,
A 240 kDa protein represents the complete beta subunit of the cyclic nucleotide-gated channel from rod photoreceptor.
1995,
Pubmed
Levitan,
Modulation of ion channels by protein phosphorylation. How the brain works.
1999,
Pubmed
Liman,
A second subunit of the olfactory cyclic nucleotide-gated channel confers high sensitivity to cAMP.
1994,
Pubmed
,
Xenbase
Liu,
Calcium-calmodulin modulation of the olfactory cyclic nucleotide-gated cation channel.
1994,
Pubmed
Liu,
Constraining ligand-binding site stoichiometry suggests that a cyclic nucleotide-gated channel is composed of two functional dimers.
1998,
Pubmed
Molokanova,
Activity-dependent modulation of rod photoreceptor cyclic nucleotide-gated channels mediated by phosphorylation of a specific tyrosine residue.
1999,
Pubmed
,
Xenbase
Molokanova,
Modulation of rod photoreceptor cyclic nucleotide-gated channels by tyrosine phosphorylation.
1997,
Pubmed
,
Xenbase
Molokanova,
Noncatalytic inhibition of cyclic nucleotide-gated channels by tyrosine kinase induced by genistein.
1999,
Pubmed
,
Xenbase
Müller,
Phosphorylation of mammalian olfactory cyclic nucleotide-gated channels increases ligand sensitivity.
1998,
Pubmed
Rehm,
Dendrotoxin-binding brain membrane protein displays a K+ channel activity that is stimulated by both cAMP-dependent and endogenous phosphorylations.
1989,
Pubmed
Reinhart,
Kinase and phosphatase activities intimately associated with a reconstituted calcium-dependent potassium channel.
1995,
Pubmed
Ruiz,
The single-channel dose-response relation is consistently steep for rod cyclic nucleotide-gated channels: implications for the interpretation of macroscopic dose-response relations.
1999,
Pubmed
,
Xenbase
Sautter,
An isoform of the rod photoreceptor cyclic nucleotide-gated channel beta subunit expressed in olfactory neurons.
1998,
Pubmed
Shammat,
Stoichiometry and arrangement of subunits in rod cyclic nucleotide-gated channels.
1999,
Pubmed
,
Xenbase
Shapiro,
Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels.
1998,
Pubmed
,
Xenbase
Sheng,
Ion channel targeting in neurons.
1997,
Pubmed
Tsai,
Receptor protein tyrosine phosphatase alpha participates in the m1 muscarinic acetylcholine receptor-dependent regulation of Kv1.2 channel activity.
1999,
Pubmed
,
Xenbase
Wiesner,
Cyclic nucleotide-gated channels on the flagellum control Ca2+ entry into sperm.
1998,
Pubmed
Wilson,
Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities.
1998,
Pubmed
Yu,
NMDA channel regulation by channel-associated protein tyrosine kinase Src.
1997,
Pubmed