Halogen substituents on the aromatic moiety of the tetracaine scaffold improve potency of cyclic nucleotide-gated channel block.

A series of new tetracaine derivatives with substituents on the aromatic ring was prepared and evaluated for block of retinal rod cyclic nucleotide-gated (CNG) channels. Aromatic substitutions had little effect starting with the basic tetracaine scaffold, but electron-withdrawing substituents significantly improved the blocking potency of an octyl-tail derivative of tetracaine. In particular, halogen substitutions at either the 2- or 3-position on the ring resulted in compounds that were up to eight-fold more potent than the parent octyl-tail derivative and up to 50-fold more potent than tetracaine.

Cyclic nucleotide-gated channel block by hydrolysis-resistant tetracaine derivatives.

To meet a pressing need for better cyclic nucleotide-gated (CNG) channel antagonists, we have increased the biological stability of tetracaine-based blockers by synthesizing amide and thioamide linkage substitutions of tetracaine (1) and a higher affinity octyl tail derivative (5). We report the apparent K(D) values, the mechanism of block, and the in vitro hydrolysis rates for these compounds. The ester linkage substitutions did not adversely affect CNG channel block; unexpectedly, thioamide substitution in 1 (compound 8) improved block significantly. Furthermore, the ester linkage substitutions did not appear to affect the mechanism of block in terms of the strong state preference for closed channels. All ester substituted compounds, especially the thioamide substitutions, were more resistant to hydrolysis by serum cholinesterase than their ester counterparts. These findings have implications for dissecting the physiological roles of CNG channels, treating certain forms of retinal degeneration, and possibly the current clinical uses of compound 1.

Block of cyclic nucleotide-gated channels by tetracaine derivatives: role of apolar interactions at two distinct locations.

A series of new tetracaine derivatives was synthesized to explore the effects of hydrophobic character on blockade of cyclic nucleotide-gated (CNG) channels. Increasing the hydrophobicity at either of two positions on the tetracaine scaffold, the tertiary amine or the butyl tail, yields blockers with increased potency. However, shape also plays an important role. While gradual increases in length of the butyl tail lead to increased potency, substitution of the butyl tail with branched alkyl or cyclic groups is deleterious.

Novel N7- and N1-substituted cGMP derivatives are potent activators of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels, key players in olfactory and visual signal transduction, generate electrical responses to odorant- and light-induced changes in cyclic nucleotide concentration. Previous work suggests that substitutions are tolerated solely at the C8 position on the purine ring of cGMP. Our studies with C8, 2'-OH, and 2-NH2-modified cGMP derivatives support this assertion. To gain further insight into determinants important for CNG channel binding and activation, we targeted previously unexplored positions. Modifications at N7 of 8-SH-cGMP (6) are well tolerated by olfactory and retinal rod CNG channels. Toleration of a very large substituent, a 3400 molecular weight PEG, at either N7 or C8 argues for broad accommodation at these positions in the binding site. Modification at N1 of cGMP reduces the apparent affinity for the channel; however, when combined with 8-parachlorophenylthio derivatization, the resulting cGMP analogue is more potent than cGMP itself. These studies establish the N7 and N1 positions of cGMP as targets for modification in the design of novel CNG channel agonists.

Cellular mechanisms underlying prostaglandin-induced transient cAMP signals near the plasma membrane of HEK-293 cells.

We have previously used cyclic nucleotide-gated (CNG) channels as sensors to measure cAMP signals in human embryonic kidney (HEK)-293 cells. We found that prostaglandin E(1) (PGE(1)) triggered transient increases in cAMP concentration near the plasma membrane, whereas total cAMP levels rose to a steady plateau over the same time course. In addition, we presented evidence that the decline in the near-membrane cAMP levels was due primarily to a PGE(1)-induced stimulation of phosphodiesterase (PDE) activity, and that the differences between near-membrane and total cAMP levels were largely due to diffusional barriers and differential PDE activity. Here, we examine the mechanisms regulating transient, near-membrane cAMP signals. We observed that 5-min stimulation of HEK-293 cells with prostaglandins triggered a two- to threefold increase in PDE4 activity. Extracellular application of H89 (a PKA inhibitor) inhibited stimulation of PDE4 activity. Similarly, when we used CNG channels to monitor cAMP signals we found that both extracellular and intracellular (via the whole-cell patch pipette) application of H89, or the highly selective PKA inhibitor, PKI, prevented the decline in prostaglandin-induced responses. Following pretreatment with rolipram (a PDE4 inhibitor), H89 had little or no effect on near-membrane or total cAMP levels. Furthermore, disrupting the subcellular localization of PKA with the A-kinase anchoring protein (AKAP) disruptor Ht31 prevented the decline in the transient response. Based on these data we developed a plausible kinetic model that describes prostaglandin-induced cAMP signals. This model has allowed us to quantitatively demonstrate the importance of PKA-mediated stimulation of PDE4 activity in shaping near-membrane cAMP signals.

The pharmacology of cyclic nucleotide-gated channels: emerging from the darkness.

Cyclic nucleotide-gated (CNG) ion channels play a central role in vision and olfaction, generating the electrical responses to light in photoreceptors and to odorants in olfactory receptors. These channels have been detected in many other tissues where their functions are largely unclear. The use of gene knockouts and other methods have yielded some information, but there is a pressing need for potent and specific pharmacological agents directed at CNG channels. To date there has been very little systematic effort in this direction - most of what can be termed CNG channel pharmacology arose from testing reagents known to target protein kinases or other ion channels, or by accident when researchers were investigating other intracellular pathways that may regulate the activity of CNG channels. Predictably, these studies have not produced selective agents. However, taking advantage of emerging structural information and the increasing knowledge of the biophysical properties of these channels, some promising compounds and strategies have begun to emerge. In this review we discuss progress on two fronts, cyclic nucleotide analogs as both activators and competitive inhibitors, and inhibitors that target the pore or gating machinery of the channel. We also discuss the potential of these compounds for treating certain forms of retinal degeneration.

Interplay between PIP3 and calmodulin regulation of olfactory cyclic nucleotide-gated channels.

Phosphatidylinositol-3,4,5-trisphosphate (PIP3) has been proposed to modulate the odorant sensitivity of olfactory sensory neurons by inhibiting activation of cyclic nucleotide-gated (CNG) channels in the cilia. When applied to the intracellular face of excised patches, PIP3 has been shown to inhibit activation of heteromeric olfactory CNG channels, composed of CNGA2, CNGA4, and CNGB1b subunits, and homomeric CNGA2 channels. In contrast, we discovered that channels formed by CNGA3 subunits from cone photoreceptors were unaffected by PIP3. Using chimeric channels and a deletion mutant, we determined that residues 61-90 within the N terminus of CNGA2 are necessary for PIP3 regulation, and a biochemical "pulldown" assay suggests that PIP3 directly binds this region. The N terminus of CNGA2 contains a previously identified calcium-calmodulin (Ca2+/CaM)-binding domain (residues 68-81) that mediates Ca2+/CaM inhibition of homomeric CNGA2 channels but is functionally silent in heteromeric channels. We discovered, however, that this region is required for PIP3 regulation of both homomeric and heteromeric channels. Furthermore, PIP3 occluded the action of Ca2+/CaM on both homomeric and heteromeric channels, in part by blocking Ca2+/CaM binding. Our results establish the importance of the CNGA2 N terminus for PIP3 inhibition of olfactory CNG channels and suggest that PIP3 inhibits channel activation by disrupting an autoexcitatory interaction between the N and C termini of adjacent subunits. By dramatically suppressing channel currents, PIP3 may generate a shift in odorant sensitivity that does not require prior channel activity.

Modifications to the tetracaine scaffold produce cyclic nucleotide-gated channel blockers with widely varying efficacies.

Five new tetracaine analogues were synthesized and evaluated for potency of blockade of cyclic nucleotide-gated channels relative to a multiply charged tetracaine analogue described previously. Increased positive charge at the tertiary amine end of tetracaine results in higher potency and voltage dependence of block. Modifications that reduce the hydrophobic character at the butyl tail are deleterious to block. The tetracaine analogues described here have apparent affinities for CNGA1 channels that vary over nearly 8 orders of magnitude.

High-resolution measurements of cyclic adenosine monophosphate signals in 3D microdomains.

A large number of hormones, neurotransmitters, and odorants exert their effects on cells by triggering changes in intracellular levels of cyclic adenosine monophosphate (cAMP). Although the effector proteins that bind cAMP have been identified, it is not known how this single messenger can differentially regulate the activities of hundreds of cellular proteins. It has been clear, for some time, that compartmentation of cAMP signals must be taking place, but the physical basis for compartmentation and the nature of local cAMP signals are mostly unknown. We present here a high-resolution method for measuring cAMP signals near the membrane in single cells. Cyclic nucleotide-gated (CNG) ion channels from olfactory receptor neurons have been genetically modified to improve their cAMP-sensing properties. We outline how these channels can be used in electrophysiological experiments to measure accurately changes in cAMP concentration near the membrane, where most adenylyl cyclases reside. We also describe how the method has been employed to dissect the roles of diffusion barriers and differential phosphodiesterase activity in creating distinct cAMP signals. This approach has much greater spatial and temporal resolution than other methods for measuring cAMP and should help to unravel the complexities of signaling by this ubiquitous messenger.

High-throughput screening of phosphodiesterase activity in living cells.

Phosphodiesterases (PDEs) hydrolyze the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine 5'-monophosphate (cGMP) and play a crucial role in the termination and spatial segregation of cyclic nucleotide signals. Despite a wealth of molecular information, very little is known about how PDEs regulate cAMP and cGMP signals in living cells because conventional methods lack the necessary spatial and temporal resolution. We present here a sensitive optical method for monitoring cAMP levels and PDE activity near the membrane, using cyclic nucleotide-gated (CNG) ion channels as sensors. These channels are directly opened by the binding of cyclic nucleotides and allow cations to cross the membrane. The olfactory channel A subunit (CNGA2) has been genetically modified to improve its cAMP sensitivity and specificity. Channel activity is assessed by measuring Ca2+ influx using standard fluorometric techniques. In addition to studying PDEs in their native setting, the approach should be particularly useful in high-throughput screening assays to test for compounds that affect PDE activity, as well as the activities of the many G protein-coupled receptors that cause changes in intracellular cAMP.

Functional role of lipid raft microdomains in cyclic nucleotide-gated channel activation.

Cyclic nucleotide-gated (CNG) channels are the primary targets of light- and odorant-induced signaling in photoreceptors and olfactory sensory neurons. Compartmentalized cyclic nucleotide signaling is necessary to ensure rapid and efficient activation of these nonselective cation channels. However, relatively little is known about the subcellular localization of CNG channels or the mechanisms of their membrane partitioning. Lipid raft domains are specialized membrane microdomains rich in cholesterol and sphingolipids that have been implicated in the organization of many membrane-associated signaling pathways. Herein, we report that the alpha subunit of the olfactory CNG channel, CNGA2, associates with lipid rafts in heterologous expression systems and in rat olfactory epithelium. However, CNGA2 does not directly bind caveolin, and its membrane localization overlaps only slightly with that of caveolin at the surface of human embryonic kidney (HEK) 293 cells. To test for a possible functional role of lipid raft association, we treated HEK 293 cells with the cholesterol-depleting agent, methyl-beta-cyclodextrin. Cholesterol depletion abolished prostaglandin E1-stimulated CNGA2 channel activity in intact cells. Recordings from membrane patches excised from CNGA2-expressing HEK 293 cells revealed that cholesterol depletion dramatically reduced the apparent affinity of homomeric CNGA2 channels for cAMP but only slightly reduced the maximal current. Our results show that olfactory CNG channels target to lipid rafts and that disruption of lipid raft microdomains dramatically alters the function of CNGA2 channels.

Resolution of cAMP signals in three-dimensional microdomains using novel, real-time sensors.

A large number of hormones, neurotransmitters, and odorants alter cellular behavior by triggering changes in intracellular levels of cAMP. Although the effector proteins that bind cAMP have been identified, it is not known how this one messenger can differentially regulate the activities of hundreds of cellular proteins. The spatial and temporal nature of cAMP signals and, thus, their information content remain largely unknown. We present here a high-resolution method for measuring cAMP signals near the plasma membrane in single cells. Cyclic nucleotide-gated (CNG) ion channels from olfactory receptor neurons have been genetically modified to improve their cAMP-sensing properties. We show how these channels can be used in electrophysiological experiments to accurately measure changes in cAMP concentration near the membrane, where most adenylyl cyclases reside. We have found in several cell types (both excitable and nonexcitable) that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is severely hindered. We also show that a uniform extracellular stimulus can initiate very distinct cAMP signals within different compartments of a simple, nonexcitable cell. Analysis of compartmental models indicates that diffusional restrictions between microdomains (near the membrane) and the cytosol, as well as differential regulation of phosphodiesterase activity, are necessary to explain such distinct signals. Using modified CNG channels as sensors has much greater spatial and temporal resolution than other methods for measuring cAMP, and should help to unravel the complexities of signaling by this ubiquitous messenger. Cyclic AMP (cAMP), the prototypical second messenger, regulates a wide variety of cellular processes. Changes in cAMP concentration transmit information to downstream effectors including protein kinase A (PKA), cyclic nucleotide-gated (CNG) channels, hyperpolarization activated (Ih) channels, and Epac. However, it is largely unclear how differential regulation of cellular targets occurs. The concept of compartmentation emerged over 20 years ago in studies of cardiac myocytes, to help explain how a variety of extracellular stimuli that primarily act through cAMP can have very different downstream effects on the cell. The basis for compartmentation, and indeed, the nature of cAMP signals themselves, have remained mysteries. To understand how these signals function within the cell it is important to answer the following questions: (i) How are cAMP signals localized? (ii) What are the kinetics of cAMP signals in localized domains? and (iii) What information is contained in the amplitude and frequency of cAMP signals? We describe here a high-resolution method for measuring cAMP signals near the plasma membrane, using modified cyclic nucleotide-gated ion channels. This approach was inspired by the field of retinal phototransduction, the best-studied second messenger signaling system, in which elegant biochemical studies have been complemented by real-time measurements of cGMP signals using endogenous cyclic nucleotide-gated (CNG) channels. CNG channels are directly opened by the binding of cyclic nucleotides. They were discovered in retinal photoreceptor cells and olfactory receptor neurons, where they generate the electrical response to light and odorants. The native retinal channel is cGMP specific, while the native olfactory channel is equally sensitive to cAMP and cGMP. Native CNG channels consist of A and B subunits, both of which bind cyclic nucleotides, although most A subunits form functional channels on their own. We have modified an olfactory channel A subunit (CNGA2) to improve its sensitivity and selectivity for cAMP. Two of the findings with this approach, summarized here, are: (i) cAMP in several cell types is produced in subcellular compartments under the plasma membrane with restricted diffusional access to the bulk cytosol; and (ii) the amplitude and kinetics of cAMP signals within these compartments are distinct from those in the remainder of the cell.

A multiply charged tetracaine derivative blocks cyclic nucleotide-gated channels at subnanomolar concentrations.

Cyclic nucleotide-gated (CNG) ion channels are central participants in sensory transduction, generating the electrical response to light in retinal photoreceptors and to odorants in olfactory receptors. They are expressed in many other tissues where their specific roles in signaling remain unclear. As is true for many other ion channels, there is a paucity of specific blockers needed to dissect the contributions of these channels to cell signaling. CNG channels are members of the superfamily of voltage-gated ion channels, and the local anesthetic tetracaine is known to block CNG channels in a manner that resembles the block of voltage-gated Na(+) channels. The amine in local anesthetics interacts with the charged selectivity filter of Na(+) channels, while the aromatic ring gets stuck in the inner cavity and has hydrophobic interactions with the residues lining that region. Here we have synthesized a derivative of tetracaine, 3-[(aminopropyl)amino]-N,N-dimethyl-N-(2-[[4-(butylamino)benzoyl]oxy]ethyl)propan-1-aminium acetate (APPA-tetracaine), that contains three positively charged amines at physiological pH instead of one. This compound blocked several different CNG channels in the picomolar to nanomolar concentration range at positive membrane potentials, making it several orders of magnitude more potent than tetracaine. In contrast, significant block of Na(+) channels by APPA-tetracaine required concentrations of hundreds of nanomolar. The results suggest that the highly charged moiety of APPA-tetracaine interacts strongly with the negative charge cluster in the selectivity filter of CNG channels. We propose that a variety of potent and specific ion channel blockers could be generated by expanding on traditional blocker structures to target the selectivity filters of other channels.

Review article: cyclic AMP sensors in living cells: what signals can they actually measure?

Cyclic AMP is a ubiquitous intracellular second messenger that transmits information to several proteins including cyclic nucleotide-gated ion channels and protein kinase A (PKA). In turn, these effectors regulate such diverse cellular functions as Ca2+ influx, excitability, and gene expression, as well as cell-specific processes such as glycogenolysis and lipolysis. The enzymes known to regulate cAMP levels, adenylyl cyclase and phosphodiesterase, have been studied in detail. Unfortunately, an understanding of how information is encoded within cAMP signals has been elusive, because, until recently, methods for measuring cAMP lacked both spatial and temporal resolution. In this paper, we describe two recently developed methods for detecting cAMP levels in living cells. The first method measures fluorescence energy transfer between labeled subunits of PKA. This method is particularly useful for monitoring cellular localization of PKA activity following increases in cAMP levels. However, the slow activation and deactivation rates, the necessarily high concentrations of labeled subunits, and the redistribution of labeled subunits throughout the cell, all intrinsic to this method, limit its utility as a cAMP sensor. The second method uses genetically modified cyclic nucleotidegated channels to measure plasma membrane-localized cAMP levels in either cell populations or single cells. The rapid gating kinetics of these channels allow real-time measurement of cAMP concentrations. These methods have given us the first glimpses of cAMP signals within living cells.

Ion channels: does each subunit do something on its own?

The advent of the patch-clamp technique 25 years ago revolutionized the study of ion channels. This method also made it possible to measure the kinetic behavior of single protein molecules. The low-noise recordings of ionic currents through single channels, coupled with other cutting-edge technologies, have revealed a rich complexity of functional states that are not readily explained by simple allosteric protein models such as the popular concerted model and the sequential model. Although these models can each account for elements of ion channel function, we propose that variations or extensions of the lesser-known general allosteric model provide a more promising framework for explaining the intricate behaviors of ion channels.