Cyclic nucleotide-gated (CNG) channels conduct Na ⍣ , K ⍣ and Ca 2⍣ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca 2⍣ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non-excitable cells, co-assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca 2⍣ signaling depends on its specific Ca 2⍣ conductance, it is necessary to analyze Ca 2⍣ permeation for each individual channel type. We have analyzed Ca 2⍣ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca 2⍣ current over the physiological range of Ca 2⍣ concentrations and found that Ca 2⍣ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca 2⍣ permeation is controlled by the Ca 2⍣ -binding affinity of the intrapore cation-binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca 2⍣ signals.

In this study, we describe two splice variants of an ether-à-go-go (EAG) K+ channel cloned from bovine retina: bEAG1 and bEAG2. The bEAG2 polypeptide contains an additional insertion of 27 amino acids in the extracellular linker between transmembrane segments S3 and S4. The heterologously expressed splice variants differ in their activation kinetics and are differently modulated by extracellular Mg2+. Cooperativity of modulation by Mg2+ suggests that each subunit of the putative tetrameric channel binds a Mg2+ ion. The channels are neither permeable to Ca2+ ions nor modulated by cyclic nucleotides. In situ hybridization localizes channel transcripts to photoreceptors and retinal ganglion cells. Comparison of EAG currents with IKx, a noninactivating K+ current in the inner segment of rod photoreceptors, reveals an intriguing similarity, suggesting that EAG polypeptides are involved in the formation of Kx channels.

Photolabile compounds which rapidly release cAMP or cGMP after photolysis are widely used for in situ studies of signaling pathways inside cells. We synthesized two novel caged compounds, 4,5-dimethoxy-2-nitrobenzyl 8-Br-cAMP (caged 8-Br-cAMP) and 4,5-dimethoxy-2-nitrobenzyl 8-Br-cGMP caged 8-BR-cGMP), which respectively release the hydrolysis-resistant analogues 8-Br-cAMP and 8-Br-cGMP. Their usefulness for physiological studies was examined in a mammalian cell line expressing the cyclic nucleotide-gated (CNG) ion channel of bovine olfactory sensory neurons. The synthesis procedure resulted in diastereomeric mixtures which were chromatographically separated into the axial and equatorial isomers of caged 8-BR-cAMP and of caged 8-BR-cGMP. The axial isomers which have a higher solubility and better solvolytic stability than the equatorial forms were used for experiments with CNG channels. Flashes of UV light produced steps in the concentration of 8-Br-cGMP which activated currents through CNG channels. Concentration steps inside the cell could be calibrated precisely using the relation between the ligand concentration and the normalized current. Similar results were obtained with caged 8-Br-cAMP. Control experiments with caged cGMP showed that flash-induced currents decayed within a few minutes because photoreleased cGMP was degraded by endogenous phosphodiesterase activity. The rise time of the 8-Br-cGMP-activated whole-cell current was consistent with a bimolecular reaction between channel and ligand.

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