Cyclic nucleotide-gated (CNG) ion channels mediate sensory signal transduction in photoreceptors and olfactory cells. Structurally, CNG channels are heterotetramers composed of either two or three homologue subunits. Although it is well established that activation is a cooperative process of these subunits, it remains unknown whether the cooperativity is generated by the ligand binding, the gating, or both, and how the subunits interact. In this study, the action of homotetrameric olfactory-type CNGA2 channels was studied in inside-out membrane patches by simultaneously determining channel activation and ligand binding, using the fluorescent cGMP analogue 8-DY547-cGMP as the ligand. At concentrations of 8-DY547-cGMP < 1 microM, steady-state binding was larger than steady-state activation, whereas at higher concentrations it was smaller, generating a crossover of the steady-state relationships. Global analysis of these relationships together with multiple activation time courses following cGMP jumps showed that four ligands bind to the channels and that there is significant interaction between the binding sites. Among the binding steps, the second is most critical for channel opening: its association constant is three orders of magnitude smaller than the others and it triggers a switch from a mostly closed to a maximally open state. These results contribute to unravelling the role of the subunits in the cooperative mechanism of CNGA2 channel activation and could be of general relevance for the action of other ion channels and receptors.

HCN pacemaker channels are tetramers mediating rhythmicity in neuronal and cardiac cells. The activity of these channels is controlled by both membrane voltage and the ligand cAMP, binding to each of the four channel subunits. The molecular mechanism underlying channel activation and the relationship between the two activation stimuli are still unknown. Using patch-clamp fluorometry and a fluorescent cAMP analog, we show that full ligand-induced activation appears already with only two ligands bound to the tetrameric channel. Kinetic analysis of channel activation and ligand binding suggests direct interaction between the voltage sensor and the cyclic nucleotide-binding domain, bypassing the pore. By exploiting the duality of activation in HCN2 channels by voltage and ligand binding, we quantify the increase of the binding affinity and overall free energy for binding upon channel activation, proving thus the principle of reciprocity between ligand binding and conformational change in a receptor protein.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.

Biologically inert, photoactivatable precursors ("caged" compounds) of cyclic nucleoside monophosphates (cNMPs) are powerful tools for studying the spatiotemporal dynamics of cyclic nucleotide dependent processes. Among these compounds, (coumarin-4-yl)methyl esters of cNMPs are most useful because they show no background bioactivity, are stable to solvolysis, and can be photolyzed efficiently and extremely quickly. [1,2] Recently, we introduced [7-(diethylamino)coumarin-4-yl]methyl (DEACM) esters of cNMPs as caged compounds. [3,4] Compared with other coumarinylmethyl-caged cNMPs, the DEACM esters photorelease cNMPs with higher photosensitivity at long-wavelength irradiation (up to 436 nm), thus minimizing or even preventing damage to cellular components and chromophores by photobleaching.Herein, we describe the development of the 7-[bis(carboxymethyl)amino]-substituted coumarinylmethyl building blocks 1 and 2 (structures in Scheme 1) for the caging of phosphates and other functionalities. With the axial and the equatorial diastereomers of the {7-[bis(carboxymethyl)-amino]coumarin-4-yl}methyl (BCMACM) esters of cAMP, cGMP, 8-Br-cAMP, and 8-Br-cGMP 3-6 (Scheme 1), we present new variants of the DEACM-caged cNMPs, which maintain their favorable properties and additionally have much higher aqueous solubility by virtue of their anionic[*] Dr.