Cyclic Nucleotide–Gated Channels Colocalize with Adenylyl Cyclase in Regions of Restricted Camp Diffusion

Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca2+, cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide–gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory α subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide–gated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22–56 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide–gated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide–gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.

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In Vivo Assessment of Local Phosphodiesterase Activity Using Tailored Cyclic Nucleotide–Gated Channels as Camp Sensors

The Journal of General Physiology ◽

10.1085/jgp.118.1.63 ◽

2001 ◽

Vol 118 (1) ◽

pp. 63-78 ◽

Cited By ~ 123

Author(s):

Thomas C. Rich ◽

Tonia E. Tse ◽

Joyce G. Rohan ◽

Jerome Schaack ◽

Jeffrey W. Karpen

Keyword(s):

Adenylyl Cyclase ◽

Cyclic Nucleotide ◽

Type I ◽

Hek 293 ◽

Cyclic Nucleotide Gated Channels ◽

Type Iv ◽

Pde Inhibitors ◽

Camp Levels ◽

Hydrolysis Of

Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotide–gated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.C., K.A. Fagan, H. Nakata, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. J. Gen. Physiol. 116:147–161). To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by Ca2+-calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced Ca2+ influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the KI of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high Km for cAMP, whereas PDE type I has a low Km for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability.

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Cyclic Nucleotide–Gated Channels

The Journal of General Physiology ◽

10.1085/jgp.114.3.377 ◽

1999 ◽

Vol 114 (3) ◽

pp. 377-392 ◽

Cited By ~ 56

Author(s):

Andrea Becchetti ◽

Katia Gamel ◽

Vincent Torre

Keyword(s):

Plasma Membrane ◽

Cyclic Nucleotide ◽

Ion Selectivity ◽

Xenopus Laevis Oocytes ◽

Gating Currents ◽

Α Subunit ◽

Channel Pore ◽

Cyclic Nucleotide Gated Channels ◽

Internal Side ◽

Inside Out

In voltage- and cyclic nucleotide–gated ion channels, the amino-acid loop that connects the S5 and S6 transmembrane domains, is a major component of the channel pore. It determines ion selectivity and participates in gating. In the α subunit of cyclic nucleotide–gated channels from bovine rod, the pore loop is formed by the residues R345–S371, here called R1-S27. These 24 residues were mutated one by one into a cysteine. Mutant channels were expressed in Xenopus laevis oocytes and currents were recorded from excised membrane patches. The accessibility of the substituted cysteines from both sides of the plasma membrane was tested with the thiol-specific reagents 2-aminoethyl methanethiosulfonate (MTSEA) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Residues V4C, T20C, and P22C were accessible to MTSET only from the external side of the plasma membrane, and to MTSEA from both sides of the plasma membrane. The effect of MTSEA applied to the inner side of T20C and P22C was prevented by adding 10 mM cysteine to the external side of the plasma membrane. W9C was accessible to MTSET from the internal side only. L7C residue was accessible to internal MTSET, but the inhibition was partial, ∼50% when the MTS compound was applied in the absence of cGMP and 25% when it was applied in the presence of cGMP, suggesting that this residue is not located inside the pore lumen and that it changes its position during gating. Currents from T15C and T16C mutants were rapidly potentiated by intracellular MTSET. In T16C, a slower partial inhibition took place after the initial potentiation. Current from I17C progressively decayed in inside-out patches. The rundown was accelerated by inwardly applied MTSET. The accessibility results of MTSET indicate a well-defined topology of the channel pore in which residues between L7 and I17 are inwardly accessible, residue G18 and E19 form the narrowest section of the pore, and T20, P21, P22 and V4 are outwardly accessible.

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Access of Quaternary Ammonium Blockers to the Internal Pore of Cyclic Nucleotide-gated Channels: Implications for the Location of the Gate

The Journal of General Physiology ◽

10.1085/jgp.200509440 ◽

2006 ◽

Vol 127 (5) ◽

pp. 481-494 ◽

Cited By ~ 29

Author(s):

Jorge E. Contreras ◽

Miguel Holmgren

Keyword(s):

Binding Site ◽

Quaternary Ammonium ◽

Cyclic Nucleotide ◽

Single Channel ◽

Dependent Manner ◽

Receptor Binding Site ◽

Α Subunit ◽

Cyclic Nucleotide Gated Channels ◽

Closed State ◽

Kv Channels

Cyclic nucleotide-gated (CNG) channels play important roles in the transduction of visual and olfactory information by sensing changes in the intracellular concentration of cyclic nucleotides. We have investigated the interactions between intracellularly applied quaternary ammonium (QA) ions and the α subunit of rod cyclic nucleotide-gated channels. We have used a family of alkyl-triethylammonium derivatives in which the length of one chain is altered. These QA derivatives blocked the permeation pathway of CNG channels in a concentration- and voltage-dependent manner. For QA compounds with tails longer than six methylene groups, increasing the length of the chain resulted in higher apparent affinities of ∼1.2 RT per methylene group added, which is consistent with the presence of a hydrophobic pocket within the intracellular mouth of the channel that serves as part of the receptor binding site. At the single channel level, decyltriethyl ammonium (C10-TEA) ions did not change the unitary conductance but they did reduce the apparent mean open time, suggesting that the blocker binds to open channels. We provide four lines of evidence suggesting that QA ions can also bind to closed channels: (1) the extent of C10-TEA blockade at subsaturating [cGMP] was larger than at saturating agonist concentration, (2) under saturating concentrations of cGMP, cIMP, or cAMP, blockade levels were inversely correlated with the maximal probability of opening achieved by each agonist, (3) in the closed state, MTS reagents of comparable sizes to QA ions were able to modify V391C in the inner vestibule of the channel, and (4) in the closed state, C10-TEA was able to slow the Cd2+ inhibition observed in V391C channels. These results are in stark contrast to the well-established QA blockade mechanism in Kv channels, where these compounds can only access the inner vestibule in the open state because the gate that opens and closes the channel is located cytoplasmically with respect to the binding site of QA ions. Therefore, in the context of Kv channels, our observations suggest that the regions involved in opening and closing the permeation pathways in these two types of channels are different.

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Cyclic Nucleotide-gated Channels on the Flagellum Control Ca2+ Entry into Sperm

The Journal of Cell Biology ◽

10.1083/jcb.142.2.473 ◽

1998 ◽

Vol 142 (2) ◽

pp. 473-484 ◽

Cited By ~ 128

Author(s):

Burkhard Wiesner ◽

Jocelyn Weiner ◽

Ralf Middendorff ◽

Volker Hagen ◽

U. Benjamin Kaupp ◽

...

Keyword(s):

Cyclic Nucleotide ◽

Photoreceptor Cells ◽

Structural Basis ◽

Α Subunit ◽

Cyclic Nucleotide Gated Channels ◽

Bending Waves ◽

Mammalian Sperm ◽

Vertebrate Photoreceptor ◽

Α And Β Subunits ◽

Principal Piece

Cyclic nucleotide-gated (CNG) channels are key elements of cGMP- and cAMP-signaling pathways in vertebrate photoreceptor cells and in olfactory sensory neurons, respectively. These channels form heterooligomeric complexes composed of at least two distinct subunits (α and β). The α subunit of cone photoreceptors is also present in mammalian sperm. Here we identify one short and several long less abundant transcripts of β subunits in testis. The α and β subunits are expressed in a characteristic temporal and spatial pattern in sperm and precursor cells. In mature sperm, the α subunit is observed along the entire flagellum, whereas the short β subunit is restricted to the principal piece of the flagellum. These findings suggest that different forms of CNG channels coexist in the flagellum. Confocal microscopy in conjunction with the Ca2+ indicator Fluo-3 shows that the CNG channels serve as a Ca2+ entry pathway that responds more sensitively to cGMP than to cAMP. Assuming that CNG channel subtypes differ in their Ca2+ permeability, dissimilar localization of α and β subunits may give rise to a pattern of Ca2+ microdomains along the flagellum, thereby providing the structural basis for control of flagellar bending waves.

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Estimating the magnitude of near-membrane PDE4 activity in living cells

AJP Cell Physiology ◽

10.1152/ajpcell.00090.2015 ◽

2015 ◽

Vol 309 (6) ◽

pp. C415-C424 ◽

Cited By ~ 7

Author(s):

Wenkuan Xin ◽

Wei P. Feinstein ◽

Andrea L. Britain ◽

Cristhiaan D. Ochoa ◽

Bing Zhu ◽

...

Keyword(s):

Enzyme Activity ◽

Plasma Membrane ◽

Cyclic Nucleotide ◽

Single Cells ◽

Cellular Functions ◽

Spatial Spread ◽

Cyclic Nucleotide Gated Channels ◽

Subcellular Compartments ◽

Camp Hydrolysis ◽

Camp Levels

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments.

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Novel mutations in the gene for α-subunit of retinal cone cyclic nucleotide-gated channels in a Japanese patient with congenital achromatopsia

Japanese Journal of Ophthalmology ◽

10.1007/s10384-016-0424-6 ◽

2016 ◽

Vol 60 (3) ◽

pp. 187-197 ◽

Cited By ~ 6

Author(s):

Kazuki Kuniyoshi ◽

Sanae Muraki-Oda ◽

Hisao Ueyama ◽

Futoshi Toyoda ◽

Hiroyuki Sakuramoto ◽

...

Keyword(s):

Cyclic Nucleotide ◽

Japanese Patient ◽

Novel Mutations ◽

Α Subunit ◽

Cyclic Nucleotide Gated Channels ◽

Retinal Cone

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A Point Mutation in the Pore Region Alters Gating, Ca2+Blockage, and Permeation of Olfactory Cyclic Nucleotide–Gated Channels

The Journal of General Physiology ◽

10.1085/jgp.116.3.311 ◽

2000 ◽

Vol 116 (3) ◽

pp. 311-326 ◽

Cited By ~ 25

Author(s):

Paola Gavazzo ◽

Cristiana Picco ◽

Elisabeth Eismann ◽

U. Benjamin Kaupp ◽

Anna Menini

Keyword(s):

Cyclic Nucleotide ◽

Single Channel ◽

Channel Gating ◽

Cation Binding ◽

Xenopus Laevis Oocytes ◽

Wild Type ◽

Pore Region ◽

Α Subunit ◽

Cyclic Nucleotide Gated Channels ◽

Cation Binding Site

Upon stimulation by odorants, Ca2+ and Na+ enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide–gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the α subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide–gated channels play a similar role. We studied the corresponding glutamate (E340) of the α subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca2+ and Mg2+. E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca2+ and Mg2+ powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca2+ relative to Na+ was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca2+ and Mg2+, and affects channel gating by cAMP more than by cGMP.

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