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Interactions of cyclic nucleotide-gated channel subunits and protein tyrosine kinase probed with genistein.

Molokanova E, Savchenko A, Kramer RH - J. Gen. Physiol. (2000)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, FL 33101, USA.

ABSTRACT
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.

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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.
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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.

Mentions: Native CNG channels differ in their sensitivity to genistein inhibition (Fig. 3). Genistein has a much larger effect on CNG channels from rods and cones than on channels from olfactory receptor neurons. Maximal inhibition of closed channels by saturating concentrations of genistein (500 μM) was 61 ± 8% (n = 9) for rod, 50 ± 3% (n = 8) for cone, and 28 ± 4% (n = 5) for olfactory channels (Fig. 3 A). As observed in homomeric RETα channels, genistein was less effective at inhibiting fully activated channels (Fig. 3 B) [inhibition of 31 ± 4% (n = 5) for rod, 37 ± 3% (n = 4) for cone, and 11 ± 4% (n = 3) for olfactory channels]. Moreover, for rod and cone CNG channels, fully activated channels had a higher apparent Ki for genistein than did closed channels (Fig. 3 C), again consistent with previous observations. For olfactory CNG channels, Ki values for genistein inhibition of closed and open channels were not significantly different. Not only did genistein have a smaller maximal effect on olfactory channels, but the apparent Ki for genistein was also higher than values for channels from rods or cones (Fig. 3 C).


Interactions of cyclic nucleotide-gated channel subunits and protein tyrosine kinase probed with genistein.

Molokanova E, Savchenko A, Kramer RH - J. Gen. Physiol. (2000)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2232887&req=5

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.
Mentions: Native CNG channels differ in their sensitivity to genistein inhibition (Fig. 3). Genistein has a much larger effect on CNG channels from rods and cones than on channels from olfactory receptor neurons. Maximal inhibition of closed channels by saturating concentrations of genistein (500 μM) was 61 ± 8% (n = 9) for rod, 50 ± 3% (n = 8) for cone, and 28 ± 4% (n = 5) for olfactory channels (Fig. 3 A). As observed in homomeric RETα channels, genistein was less effective at inhibiting fully activated channels (Fig. 3 B) [inhibition of 31 ± 4% (n = 5) for rod, 37 ± 3% (n = 4) for cone, and 11 ± 4% (n = 3) for olfactory channels]. Moreover, for rod and cone CNG channels, fully activated channels had a higher apparent Ki for genistein than did closed channels (Fig. 3 C), again consistent with previous observations. For olfactory CNG channels, Ki values for genistein inhibition of closed and open channels were not significantly different. Not only did genistein have a smaller maximal effect on olfactory channels, but the apparent Ki for genistein was also higher than values for channels from rods or cones (Fig. 3 C).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, FL 33101, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus