Correspondence to: Richard H. Kramer, P.O. Box 016189, University of Miami School of Medicine, Miami, FL 33101. Fax:305-243-4555 E-mail:rkramer{at}chroma.med.miami.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cGMP sensitivity of cyclic nucleotidegated (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 subunits (RET
) 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 RET
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 ß as well as
subunits. To determine the contributions of
and ß subunits to genistein inhibition, we compared the effect of genistein on native, homomeric (RET
and OLF
), and heteromeric (RET
+ß, OLF
+ß, and OLF
+RETß) CNG channels. We found that genistein only inhibits channels that contain either the RET
or the OLFß subunits. This finding, along with other observations about the maximal effect of genistein and the Hill coefficient of genistein inhibition, suggests that the RET
and OLFß subunits contain binding sites for the PTK, whereas RETß and OLF
subunits do not.
Key Words: cyclic guanosine monophosphate, protein tyrosine kinase, photoreceptor, olfactory receptor neuron
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyclic nucleotidegated (CNG)1 channels are crucial for sensory transduction in rod and cone photoreceptors and olfactory sensory neurons. The cyclic nucleotide sensitivity of CNG channels can be modulated by various intracellular signals including Ca2+/calmodulin ( subunits (RET
) can be modulated by protein tyrosine kinases (PTKs) and phosphatases (PTPs) intrinsic to the oocyte (
We have probed the interaction between the rod CNG channel and the oocyte PTK using the tyrosine kinase inhibitor genistein ( channels by triggering a noncatalytic interaction between oocyte PTKs and the channel that allosterically inhibits channel gating.
Are native CNG channels in sensory cells, like CNG channels expressed in oocytes, associated with PTKs? Our recent work shows that CNG channels in rod outer segments are subject to modulation by changes in tyrosine phosphorylation, the final step in a biochemical cascade in which neuromodulators can alter the rod light response (Savchenko, Kraft, Molokanova, Kramer, manuscript in preparation). Since genistein has been useful in probing the interaction between the cloned RET channel and oocyte PTKs, we asked whether genistein would reveal a similar interaction between native CNG channels and PTKs in sensory cells, namely rods, cones, and olfactory receptor neurons.
Both the native rod and olfactory CNG channels are heteromers containing and ß subunits (
and RETß in rods and OLF
and OLFß in olfactory neurons. Exogenous expression of
subunits alone generates functional CNG channels in oocytes. In contrast, ß subunits do not form functional channels when expressed alone. However, coexpression of
and ß subunits yields channels with properties that more closely resemble native rod and olfactory channels. To better understand how
and ß subunits of CNG channels interact with PTKs, we compared the effects of genistein on native, cloned homomeric, and cloned heteromeric channels. Our results suggest that all three native CNG channels (rod, cone, and olfactory) are indeed associated with PTKs. However, for the rod channel, the
subunit is crucial for allowing interaction with the PTK, whereas in the olfactory CNG channel, the ß subunit is crucial.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recording from Native CNG Channels
Water-phase tiger salamanders (Ambystoma tigrinum) maintained in a temperature-controlled aquarium (4°C) on a 12/12 light/dark cycle were used in experiments at the same time of day. Animals were dark-adapted for 1 h and anesthetized in an ice-cold solution containing 1 g/liter of 2-amino benzoic acid for 20 min before decapitation and removal of eyes under dim red light. Eyes were placed in physiological saline containing (mM): 155 NaCl, 2.5 KCl, 1 CaCl2, 2 MgCl2, 10 glucose, and 10 HEPES, pH 7.5. Retinas were removed and gently triturated to yield isolated rods, cones, or rod outer segments. Borosilicate glass pipettes (35 M) were filled with standard patch solution containing (mM): 115 NaCl, 5 EGTA, 1 EDTA, and 5 HEPES, pH 7.5 with NaOH, and were used to obtain excised inside-out patches from outer segments of rods and cones.
Olfactory receptor neurons were acutely dissociated from the olfactory epithelium of land-phase tiger salamanders. After dissection, the epithelium was placed for 1822 min at 21°C in a tube containing 3 ml of physiological solution [(mM)148 NaCl, 4 KCl, 1.5 CaCl2, 2 MgCl2, 10 glucose, 10 HEPES, pH 7.4] containing 48 µl of cysteine-activated papain (Worthington Pharmaceuticals). The tissue was then continuously washed for 10 min in ice cold papain-free solution and gently triturated to isolate individual olfactory receptor neurons. Patch pipettes with resistances of 1020 M were used for obtaining excised patches. Pipettes were filled with a solution containing (mM): 150 NaCl, 4 KCl, 5 EGTA, 0.2 MgCl2, 10 glucose, 10 HEPES, pH 7.4. This also served as the bath solution and cGMP perfusion solution for experiments on olfactory neurons.
Expression and Recording from CNG Channels Expressed in Xenopus Oocytes
To generate homomeric channels, a cDNA clone encoding the subunits of the bovine rod photoreceptor CNG channel (
) were filled with standard patch solution, which also served as the standard bath solution and cGMP perfusion solution. After formation of a gigaohm seal, inside-out patches were excised and the patch pipette was quickly (<30 s) placed in the outlet of a 1-mm diameter tube for application of cGMP and other agents. We used a perfusion manifold containing up to eight different solutions capable of solution changes within 50 ms. Most patches contained 100200 channels.
To generate heteromeric RET+ß channels, mRNA encoding RET
and RETß (
channels (
to RETß mRNA produced channels that were inhibited 78 and 80%, respectively, by 10 µM L-cis diltiazem. In contrast, a 1:5 ratio generated channels inhibited by 50%, suggesting that a RET
:RETß mRNA ratio of 1:10 is sufficient to saturate the RETß contribution to heteromeric channels, consistent with previous results (
+ß heteromeric channels, OLF
and OLFß (
channels [K1/2 of 9.9 ± 0.8 µM (n = 11) and 130 ± 9 µM (n = 12), respectively].
For expression of hybrid OLF+RETß channels, mRNAs encoding the two subunits were co-injected into oocytes. The presence of RETß in hybrid channels was confirmed by applying 10 µM L-cis diltiazem (
to RETß mRNA-injection ratios of 1:1.5 and 1:3 by 36.8 ± 2.9% (n = 3) and 32.8 ± 1.4% (n = 6), respectively. These results suggest that a OLF
to RETß injection ratio of 1:3 is sufficient to saturate the contribution of RETß to hybrid channels.
Analysis of Genistein Inhibition
Genistein and erbstatin (stable analogue) were purchased from Alexis Corp. and were prepared as concentrated stock solutions in DMSO. Aqueous solutions containing the final concentrations were prepared for use as needed. The final concentration of DMSO did not exceed 0.1%, which had no effect on CNG channels or their modulation. Cyclic GMP and AMP-PNP were purchased from Sigma Chemical Co. Current responses through CNG channels were obtained with an Axopatch 200A patch clamp (Axon Instruments, Inc.), digitized, stored, and later analyzed using pClamp 6.0 software. Membrane potential was held at -75 mV in all experiments. Current responses were normalized to the maximal CNG current (Imax), elicited by saturating (2 mM) cGMP. To estimate the Ki for genistein, we used a modified Hill equation: Ib/Imax = [1 - (Ib(max)/Imax)]/[1 + (Ki/B)n] + Ib(max)/Imax, where B is the concentration of blocker, Ib and Ib(max) are the currents activated by saturating cGMP in the presence of a given blocker concentration, and a saturating blocker concentration, respectively. Genistein inhibition of closed and fully activated channels was analyzed as described previously (
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genistein Inhibition of Native CNG Channels
Application of genistein on expressed homomeric rod channels (RET) stabilizes the closed state of the channel, such that preapplication of genistein slows activation upon exposure to cGMP (
= 8.5 s), while the residual current activates over the normal rapid time course (
= 20 ms). By examining these two components with different concentrations of genistein, we previously showed that the slowly activating current reflects channels inhibited by genistein, whereas the residual current reflects channels that are unaffected by genistein. Genistein also inhibits channels after they have been maximally activated by saturating cGMP (Fig 1 C), but the extent of inhibition is less than for closed channels. Hence, genistein more effectively inhibits closed than open rod channels, as was observed previously for the expressed RET
channel.
|
Genistein inhibition of the expressed RET channel is suppressed by coapplication of PTK inhibitors that have no direct effects on CNG channels (
|
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 RET 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).
|
Role of RET and RETß Subunits in Genistein Inhibition of Rod CNG Channels
While inhibition of native rod channels is qualitatively similar to that of expressed homomeric RET channels, there are striking quantitative differences. Thus, saturating genistein can completely suppress activation of closed RET
channels (inhibition of 98 ± 5%; n = 20), whereas activation of native channels is maximally reduced by only 61 ± 8% (n = 9). Fig 4 shows that the RETß subunit almost entirely accounts for this difference. Thus, heteromeric RET
+ß channels behave in a manner very similar to native rod channels, with channels in their closed state exhibiting a maximal genistein inhibition of 53 ± 5% (n = 10) (Fig 4 A). Moreover, the apparent Ki of heteromeric RET
+ß channels was 20 µM, a value much higher than for homomeric RET
channels (4 µM), but similar to that for native rod channels (19 µM) (Fig 4 B). The Hill coefficient also differs for native rod CNG channels (~1) and homomeric RET
channels (~2), but the value for heteromeric RET
+ß channels (~2) is indistinguishable from that for native channels.
|
The same is true of rod channels in their open state. Genistein inhibition of native rod channels more closely resembles inhibition of heteromeric RET+ß than of homomeric RET
channels (Fig 5 A). In the presence of saturating cGMP (2 mM), genistein inhibits native, homomeric RET
, and heteromeric RET
+ß channels by 31 ± 4% (n = 5), 67 ± 4% (n = 18), and 37 ± 3% (n = 9), respectively. Fig 5 B shows that Ki values for genistein on fully activated channels was ~51 µM for native, 82 µM for RET
, and 53 µM for RET
+ß. The Hill coefficient was similar for inhibition of each of the open channels, with values near 1 for each. However, while the Hill coefficients for inhibition of native and heteromeric RET
+ß are the same for open and closed channels (~1 for each), for homomeric RET
channels, the Hill coefficient changes from 2 when the channels are closed to 1 when the channels are fully activated.
|
The ß subunit also accounts for differences in the kinetics of genistein inhibition. Genistein inhibition of open native rod channels is relatively fast, with a time constant of 1.2 s for fully activated channels (see Fig 1 C). In contrast, the time course of genistein inhibition of open homomeric RET channels is much slower (Fig 6 A), with a time constant of 9 s. The kinetics of inhibition of heteromeric RET
+ß channels (Fig 6 B) are similar to that of native channels with a time constant of ~1 s. The kinetics of genistein inhibition are similar at a range of genistein concentrations from 25 to 1,000 µM. This suggests that the rate-limiting step in inhibition is not genistein binding, but rather the interaction between the PTK and channel subunits.
|
Role of OLF and OLFß Subunits in Genistein Inhibition of Olfactory CNG Channels
Like rod CNG channels, native olfactory CNG channels are heteromers containing OLF and OLFß subunits, in addition to a splice variant of the RETß subunit (
, like RET
, is the primary target for genistein inhibition, we examined the effect of genistein on homomeric OLF
channels. Fig 7 shows that genistein fails to inhibit both closed (A) and fully activated (B) OLF
channels, at concentrations up to 500 µM (n = 4). In contrast, heteromeric channels, formed by coexpressing OLF
and OLFß subunits were inhibited to an even greater extent than were native olfactory channels.
|
Studies on hybrid RET+OLF channels
The observation that OLFß is necessary for genistein inhibition suggests that the PTK responsible for indirectly conferring genistein sensitivity can selectively associate with OLFß, rather than with OLF subunits. Do the two RET subunits, RET
and RETß, also differ in their ability to interact with the PTK and confer genistein inhibition? To address this question, we took advantage of the observation that OLF
is apparently unable to interact with the PTK. If RETß can interact with the PTK, one might expect that hybrid heteromeric channels (OLF
+RETß) generated by coexpressing OLF
with RETß would be subject to genistein inhibition. Expression of such hybrid channels was confirmed by examining their sensitivity to L-cis diltiazem, which has a heightened affinity for blocking channels that contain the RETß subunit. Fig 8 shows that genistein has no effect on these hybrid channels, strongly suggesting that the RETß subunit does not bind to the PTK responsible for genistein inhibition. Hence we propose that genistein inhibition of rod CNG channels is mediated selectively by the interaction between the PTK and the RET
subunit.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Multimolecular Complexes between CNG Channels and PTKs
There is a growing realization that ion channels do not exist in isolation, but rather coexist with other proteins in macromolecular complexes. Protein partners of ion channels include cytoskeletal components, anchored via PDZ and other domains (
Our studies strongly suggest that CNG channels are part of a regulatory complex that contains PTKs and PTPs. The cGMP sensitivity of homomeric RET channels expressed in Xenopus oocytes increases after patch excision, owing to tyrosine dephosphorylation by PTPs (
channels.
Application of PTP and PTK inhibitors to intact rods shows that the sensitivity of native rod CNG channels is also modulated by changes in tyrosine phosphorylation (
We have used the PTK inhibitor genistein to probe the interaction between PTKs and CNG channels. In previous studies ( channels even in the absence of ATP, indicating that the inhibition does not require catalytic activity of PTKs. However, the genistein effect was suppressed by PTK inhibitors that do not directly interact with CNG channels (AMP-PNP and erbstatin), strongly suggesting that genistein does not directly bind to the channel, but rather affects channel gating indirectly by binding to a PTK. From these studies, we concluded that the PTK can allosterically influence channel gating even without catalyzing phosphorylation. In this paper, we find characteristics of genistein inhibition of native rod CNG channels that are entirely consistent with our previous results on expressed channels, supporting the notion that a similar macromolecular complex, including at least CNG channels and a PTK, must exist in rod outer segments. Since we observe at least some degree of genistein inhibition not only in rods, but also in cones and olfactory neurons, CNG channels in all of these sensory cells may occur in macromolecular complexes with PTKs. The placement of regulatory enzymes, such as PTKs and PTPs, in close apposition with substrate proteins, such as CNG channels, may be important for efficient and rapid signaling. We suggest that, in rods, a complex of CNG channels, PTKs, and perhaps PTPs may be part of a larger "transducisome," also containing other proteins involved in phototransduction (
Role of and ß Subunits in Genistein Inhibition
Since homomeric RET channels are strongly inhibited by genistein, it appears that the RET
subunit alone is sufficient for binding the PTK. For heteromeric RET
+ß channels, genistein has a lower apparent affinity, smaller maximal effect, and a smaller Hill coefficient for inhibition of closed channels. These observations are consistent with the possibility that genistein inhibition of RET
+ß channels is solely mediated by interaction between the PTK and the RET
subunit, with the RETß subunit unable to bind. However, it also possible that the PTK binds to both subunits, but in the context of the heteromeric RET
+ß channel, binding of the PTK to each subunit is less favorable. The observation that genistein has no effect on hybrid OLF
+RETß channels helps distinguish between these possibilities. Thus, the RETß subunit is unable to confer genistein sensitivity when coexpressed with OLF
subunits, which by itself is insensitive. The OLFß subunit does confer genistein sensitivity when coexpressed with OLF
, indicating that OLF
is not simply "dominant negative" for genistein inhibition. Hence, we conclude that genistein inhibition of rod channels is exclusively mediated by an interaction between the PTK and the RET
subunit. The RETß subunit does appear to alter the kinetics of this interaction (Fig 6), perhaps through an allosteric influence on RET
subunits.
The situation is different for olfactory CNG channels. Homomeric OLF channels are unaffected by genistein, whereas heteromeric OLF
+ß channels are inhibited, suggesting that the OLFß subunit, rather than the OLF
, interacts with the PTK. Hence, whereas the
subunit interacts with the PTK in rod CNG channels, the ß subunit interacts with the PTK in olfactory CNG channels. It should be noted that the nomenclature of CNG channel subunits (
vs. ß) is not based on sequence homology, but rather is based on the chronology of subunit cloning, such that the first cloned subunits were named "
" and the second named "ß." In fact, the two subunits that are unable to interact with the PTK (OLF
and RETß) share a homologous domain in the NH2 terminus that enables these subunits to interact with Ca2+/calmodulin (
and OLFß subunits share regions that allow interaction with the PTK, enabling genistein inhibition. In addition, it should be noted that native olfactory CNG channels appear to contain not only OLF
and OLFß subunits, but also a splice variant of the RETß subunit (
+ß channels (Fig 8), the lack of RETß subunit, which apparently does not bind PTK, may account for the greater degree of genistein inhibition as compared with native olfactory channels.
Stoichiometry of Interactions between PTKs and CNG Channel Subunits
Fig 9 A presents one possible mechanism for how PTKs might interact with the subunits of CNG channels. The Hill coefficient for genistein inhibition of closed homomeric RET channels is 2. If this accurately reflects the number of genistein binding sites, it would suggest that two PTKs are involved in channel inhibition. Since the homomeric channel contains four identical subunits, it is possible that each PTK simultaneously interacts with two neighboring subunits (Fig 9 A, left). Many receptor-type PTKs, such as growth factor receptors, either contain repeating domains or are dimeric proteins (
|
What accounts for the curious decrease in the value of the Hill coefficient from two to one as RET channels go from closed to open during activation? Recent studies suggest that rather than acting independently or in a completely concerted manner, the four subunits of some CNG channels may function as a pair of independent dimers. Thus, for each functional dimer, the two participating channel subunits cooperatively bind cyclic nucleotide and activate in a concerted manner (
subunits is dependent on binding of cyclic nucleotide. Hence, whereas all the RET
subunits are equivalent when the channel is closed, cGMP binding and/or channel opening triggers association of adjacent channel subunits, allowing them to function as a dimer. The minimal number of genistein molecules required to inhibit a channel containing such a pair of functional dimers is 1. This assumes that the PTK preferentially interacts "perpendicularly" with respect to the functional dimers; that is, by binding to RET
subunits on two distinct functional dimers (Fig 9 A, right). Since the PTK can bind to the channels in their closed state, binding of the PTK to two adjacent RET
subunits may actually determine which RET
subunits functionally dimerize, namely the RET
subunits that are not already joined by a PTK. In this scenario, PTK-dependent subunit linkage between subunits and cGMP-dependent functional dimerization are mutually exclusive.
In the heteromeric RET+ß channel (Fig 9 B), only the RET
subunits are capable of binding the PTK. Hence, if the channel stoichiometry is indeed RET
2, RETß2, a single PTK dimer could interact with adjacent RET
subunits, fully accounting for genistein inhibition because the RETß subunits appear to be immune to genistein inhibition. This scenario is consistent with our observed Hill coefficient for genistein inhibition of 1, and also accounts for the reduced maximal effect of genistein. Moreover, the proposed subunit arrangement is supported by recent studies by
subunits in heteromeric RET channels are adjacent rather than diagonally opposed.
The role of and ß subunits with respect to genistein inhibition is reversed in the OLF channel. Homomeric OLF
channels are unaffected by genistein, suggesting that the OLF
subunit cannot interact with the PTK (Fig 9 C). However, the OLFß subunit confers genistein sensitivity, suggesting that OLFß subunits contain a PTK binding site. The Hill coefficient of heteromeric OLF
+ß channels is one for both closed and fully activated channels, consistent with PTK spanning two functional dimers via interaction with two adjacent ß subunits (Fig 9 D).
These diagrams are speculative and are only a starting point for considering the stoichiometric relationships between PTKs and subunits of CNG channels. Estimates of stoichiometries based on Hill coefficients need to be confirmed with more direct biochemical experiments. Various factors, including heterogeneous populations of channels (
Differences in the ability of and ß subunits of rod and olfactory CNG channels to interact with PTKs has implications beyond genistein inhibition. It is likely that susceptibility to genistein inhibition is related to a channel's ability to be modulated by tyrosine phosphorylation. Thus, channels containing only subunits that are insensitive to genistein (e.g., OLF
homomers) might be insensitive to neuromodulators that signal through PTPs or PTKs, whereas channels that contain genistein-sensitive subunits would be susceptible to such neuromodulators. Biochemical and electrophysiological studies show that expression of olfactory CNG channel subunits is especially widespread, in the nervous system and elsewhere, but channels in different locations may differ in their subunit compositions (
![]() |
Footnotes |
---|
Dr. Savchenko's present address is Affymax Research Institute, Palo Alto, CA 94304.
1 Abbreviations used in this paper: AMP-PNP, adenylylimidodiphosphate; CNG, cyclic nucleotide-gated; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase.
Submitted: 31 December 1999
Revised: 31 March 2000
Accepted: 11 April 2000
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berghard, A., Buck, L.B., Liman, E.R. 1996. Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs. Proc. Natl. Acad. Sci. USA. 93:2365-2369
Biefeld, K., Jackson, M.B. 1994. Phosphorylation and dephosphorylation modulate a Ca2+-activated K+ channel in rat peptidergic nerve terminals. J. Physiol. 475:241-254[Abstract].
Bonigk, W., Bradley, J., Muller, F., Sesti, F., Boekhoff, I., Ronnett, G.V., Kaupp, U.B., Frings, S. 1999. The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits. J. Neurosci. 19:5332-5347
Bradley, J., Li, J., Davidson, N., Lester, H.A., Zinn, K. 1994. Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP. Proc. Natl. Acad. Sci. USA. 91:8890-8894[Abstract].
Chen, T.Y., Peng, Y.W., Dhallan, R.S., Ahamed, B., Reed, R.R., Yau, K.W. 1993. A new subunit of the cyclic nucleotide-gated cation channel in retinal rods. Nature. 362:764-767[Medline].
Chen, T.Y., Illing, M., Molday, L.L., Hsu, Y.T., Yau, K.-W., Molday, R.S. 1994. Subunit 2 (or beta) of retinal rod cGMP-gated cation channel is a component of the 240-kDa channel-associated protein and mediates Ca2+-calmodulin modulation. Proc. Natl. Acad. Sci. USA. 91:11757-11761
Dhallan, R.S., Yau, K.-W., Schrader, K.A., Reed, R.R. 1992. Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature. 347:184-187.
Fantl, W.F., Johnson, D.E., Williams, L.T. 1993. Signalling by receptor tyrosine kinases. Annu. Rev. Biochem. 62:453-481[Medline].
Finn, J.T., Kraytwurst, D., Schroeder, J.E., Chen, T.-Y., Reed, R.R., Yau, K.-W. 1998. Functional co-assembly among subunits of cyclic nucleotide-activated nonselective cation channels, and across species from nematode to human. Biophys. J. 74:1333-1345
Gordon, S.E., Brautigan, D.L., Zimmerman, A.L. 1992. Protein phosphatases modulate the apparent agonist affinity of the light-regulated ion channel in retinal rods. Neuron. 9:739-748[Medline].
Grunwald, M.E., Yu, W.P., Yu, H.H., Yau, K.-W. 1998. Identification of a domain on the beta-subunit of the rod cGMP-gated cation channel that mediates inhibition by calcium-calmodulin. J. Biol. Chem. 273:9148-9157
Holmes, T.C., Fadool, D.A., Ren, R., Levitan, I.B. 1996. Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain. Science. 274:2089-2091
Imoto, M., Umezawa, K., Isshiki, K., Kunimoto, S., Sawa, T., Takeuchi, T., Umezawa, H. 1987. Kinetic studies of tyrosine kinase inhibition by erbstatin. J. Antibiot. (Tokyo) 40:1471-1473[Medline].
Kaupp, U.B., Niidome, T., Tanabe, T., Terada, S., Bonigk, W., Stühmer, W., Cook, N.J., Kangawa, K., Matsuo, H., Hirode, T. et al. 1989. Primary structure and functional expression from complementary DNA of the rod photoreceptor cGMP-gated channel. Nature. 342:762-766[Medline].
Koerschen, H.G., Illing, M., Seifert, R., Sesti, F., Williams, A., Gotzes, S., Colville, C., Muller, F., Dose, A., Godde, M. et al. 1995. A 240 kDa protein represents the complete beta subunit of the cyclic nucleotide-gated channel from rod photoreceptor. Neuron. 15:627-636[Medline].
Koerschen, H.G., Beyermann, M., Muller, F., Heck, M., Vantler, M., Koch, K.W., Kellner, R., Wolfrum, U., Bode, C., Hofmann, K.P., Kaupp, U.B. 1999. Interaction of glutamic-acid-rich proteins with the cGMP signalling pathways in rod photoreceptors. Nature. 400:761-766[Medline].
Levitan, I.B. 1999. Modulation of ion channels by protein phosphorylation. Adv. Second Messenger Phosphoprotein Res 33:3-22[Medline].
Liman, E.R., Buck, L.B. 1994. A second subunit of the olfactory cyclic nucleotide-gated channel confers high sensitivity to cAMP. Neuron. 13:611-621[Medline].
Liu, D.T., Tibbs, G.R., Paoletti, P., Siegelbaum, S.A. 1998. Constraining ligand-binding site stoichiometry suggests that a cyclic nucleotide-gated channel is composed of two functional dimers. Neuron. 21:235-248[Medline].
Liu, M., Chen, T.-Y., Ahamed, B., Li, J., Yau, K.-W. 1994. Calcium-calmodulin modulation of the olfactory cyclic nucleotide-gated cation channel. Science 266:1348-1354[Medline].
Molokanova, E., Trivedi, B., Savchenko, A., Kramer, R.H. 1997. Modulation of rod photoreceptor cyclic nucleotide-gated channels by tyrosine phosphorylation. J. Neurosci. 17:9068-9076
Molokanova, E., Savchenko, A., Kramer, R.H. 1999a. Noncatalytic inhibition of cyclic-nucleotidegated channels by tyrosine kinase induced by genistein. J. Gen. Physiol 113:45-56
Molokanova, E., Maddox, F., Luetje, C.W., Kramer, R.H. 1999b. Activity-dependent modulation of rod photoreceptor cyclic nucleotide-gated channels mediated by phosphorylation of a specific tyrosine residue. J. Neurosci. 19:4786-4795
Muller, F., Bonigk, W., Sesti, F., Frings, S. 1998. Phosphorylation of mammalian olfactory cyclic nucleotide-gated channels increases ligand sensitivity. J. Neurosci 18:164-173
Rehm, H., Pelzer, S., Cochet, C., Chambaz, E., Tempel, B.L., Trautwein, W., Pelzer, D., Lazdunski, M. 1989. Dendrotoxin-binding brain membrane protein displays a K+ channel activity that is stimulated by both cAMP-dependent and endogenous phosphorylations. Biochemistry. 28:6455-6460[Medline].
Reinhart, P.H., Levitan, I.B. 1995. Kinase and phosphatase activities intimately associated with a reconstituted calcium-dependent potassium channel. J. Neurosci. 15:4572-4579[Abstract].
Ruiz, M., Brown, R.L., He, Y., Haley, T.L., Karpen, J.W. 1999. The single-channel doseresponse relation is consistently steep for rod cyclic nucleotide-gated channels: implications for the interpretation of macroscopic doseresponse relations. Biochemistry. 38:10642-10648[Medline].
Sautter, A., Zong, X., Hofmann, F., Biel, M. 1998. An isoform of the rod photoreceptor cyclic nucleotide-gated channel beta subunit expressed in olfactory neurons. Proc. Natl. Acad. Sci. USA. 95:4696-4701
Shammat, I.M., Gordon, S.E. 1999. Stoichiometry and arrangement of subunits in rod cyclic nucleotide-gated channels. Neuron. 23:809-819[Medline].
Shapiro, M.S., Zagotta, W.N. 1998. Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated channels. Proc. Natl. Acad. Sci. USA. 95:14546-14551
Sheng, M., Wyszynski, M. 1997. Ion channel targeting in neurons. Bioessays. 19:847-853[Medline].
Tsai, W., Morielli, A.D., Cachero, T.G., Peralta, E.G. 1999. Receptor protein tyrosine phosphatase alpha participates in the M1 muscarinic acetylcholine receptor-dependent regulation of Kv1.2 channel activity. EMBO (Eur. Mol. Biol. Organ.) J. 18:109-118
Wiesner, B., Weiner, J., Middendorff, R., Hagen, V., Kaupp, U.B., Weyand, I. 1998. Cyclic nucleotidegated channels on the flagellum control Ca2+ entry into sperm. J. Cell. Biol. 142:473-484
Wilson, G.F., Magoski, N.S., Kaczmarek, L.K. 1998. Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities. Proc. Natl. Acad. Sci. USA. 95:10938-10943
Yu, X.M., Askalan, R., Keil, G.J., Salter, M.W. 1997. NMDA channel regulation by channel-associated protein tyrosine kinase Src. Science. 275:674-678