Jeffrey W. Karpen

A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell

cAMP, the classical second messenger, regulates many diverse cellular functions. The primary effector of cAMP signals, protein kinase A, differentially phosphorylates hundreds of cellular targets. Little is known, however, about the spatial and temporal nature of cAMP signals and their information content. Thus, it is largely unclear how cAMP, in response to different stimuli, orchestrates such a wide variety of cellular responses. Previously, we presented evidence that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is hindered. Here we report that a uniform extracellular stimulus initiates distinct cAMP signals within different cellular compartments. By using cyclic nucleotide-gated ion channels engineered as cAMP biosensors, we found that prostaglandin E1 stimulation of human embryonic kidney cells caused a transient increase in cAMP concentration near the membrane. Interestingly, in the same time frame, the total cellular cAMP rose to a steady level. The decline in cAMP levels near the membrane was prevented by pretreatment with phosphodiesterase inhibitors. These data demonstrate that spatially and temporally distinct cAMP signals can coexist within simple cells.

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.

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.

Direct spectrophotometric detection of cation flux in membrane vesicles: stopped-flow measurements of acetylcholine-receptor-mediated ion flux.

The development of a spectrophotometric stopped-flow method to measure ion flux in membrane vesicles in the millisecond to minute time region is described in detail. The technique is based on fluorescence quenching of an entrapped fluorophore (anthracene-1,5-disulfonic acid) by Cs+. The method has been applied to the measurement of acetylcholine-receptor-mediated ion flux in membrane vesicles prepared from the electric organs of both Electrophorus electricus and Torpedo californica. The method is applicable to any vesicle system in which Cs+ can substitute for either Na+ or K+. Loading of vesicles with the fluorescent dye is accomplished using the routine procedure for making the vesicles. The dye-loaded vesicles can be stored in liquid nitrogen before use. Neither the dye-loading procedure nor the presence of Cs+ changes the permeability of the membrane to ions, allowing ion-translocation measurements to be made in the millisecond to minute time region. The stopped-flow design presented allows two sequential mixings of solutions. The relationship between fluorescence quenching and ion flux as well as the interpretation of the ion flux data is described. It is shown that the data obtained with stopped-flow and Cs+ is identical to data obtained previously using a quench-flow technique and 86Rb+. The advantages of the present method over the quench-flow technique and a similar stopped-flow technique developed previously based on T1+ are described in detail.

Spectrophotometric detection of monovalent cation flux in cells: Fluorescence microscope measurement of acetylcholine receptor-mediated ion flux in PC-12 cells

A new and convenient spectroscopic method for measuring monovalent cation flux in cells is described. The technique is based on fluorescence quenching of an entrapped fluorophore (anthracene-1,5-dicarboxylic acid) by Cs+. A conventional fluorescence microscope can be used to measure the Cs+ flux. The usefulness of the technique is illustrated by measurement of acetylcholine receptor-mediated Cs+ flux in PC-12 cells, a sympathetic neuronal cell line. The results are the same as those obtained when radioactive tracer ions were used. The technique is applicable to any transmembrane process in which Cs+ can substitute for either Na+ or K+. The method has been developed to identify the different neurotransmitter receptors that control the translocation of monovalent cations and to locate the cells in central nervous system cell preparations that contain these receptors. The advantage of an optical method over tracer ion methods for biochemical and pharmacological studies of transmembrane processes in cells is described.

Acetylcholine receptor-controlled ion translocation. A comparison of the effects of suberyldicholine, carbamoylcholine, and acetylcholine

Summary The effect of suberyldicholine concentration (0.5 to 30 μM) on acetylcholine receptor-controlled ion flux was measured in membrane vesicles prepared from E. electricus . Both a quench flow apparatus and a stopped flow technique were used. The integrated rate equation, based on a minimum model that relates ligand binding to ion translocation, accounts for the effects of acetylcholine, carbamoylcholine, and suberyldicholine over a wide range of concentrations. The different maximum influx rates obtained with these 3 ligands are accounted for by their effect on the equilibrium between receptor: ligand complexes in the open and closed channel forms. The constants for these equilibria have been determined.

Phencyclidine inhibition of the acetylcholine receptor: Measurement of cation flux in a sympathetic neuronal cell line using 22Na+ and spectroscopic detection of Cs+

The site of action of phencyclidine, a powerful and increasingly abused drug, in sympathetic nerve cells has not previously been identified. Here it is demonstrated that phencyclidine is a powerful, noncompetitive inhibitor of the nicotinic acetylcholine receptor in a sympathetic nerve cell line, PC-12. In the presence of 1 mM carbamoylcholine the rate of the receptor-controlled influx of 22Na+ is reduced by a factor of 2 by 0.7 microM phencyclidine. Increasing concentrations of carbamoylcholine cannot reverse the inhibitory effect of the drug. Both the transmission of electrical signals between nerve cells and the secretion of catecholamines in the PC-12 cell line depend on the receptor-controlled ion flux. Thus phencyclidine interferes with at least two specific, physiologically important functions of these nerve cells. A new spectroscopic method has been developed to measure cation flux in cells. It is shown that this method can replace measurements of tracer ion flux.

Ion channel structure and the promise of bacteria: cyclic nucleotide-gated channels in the queue.

To date it has proved very difficult to express eukaryotic channels in Escherichia coli or otherwise obtain enough of these proteins for crystallization. So, unbeknownst to bacteria, they have been making a major contribution to ion channel research since 1998. So much so that everyone in the ion