Distinct Structural Determinants of Efficacy and Sensitivity in the Ligand-binding Domain of Cyclic Nucleotide-gated Channels

Abstract

Cyclic nucleotide-gated (CNG) channels open in response to direct binding of cyclic nucleotide messengers. Every subunit in a tetrameric CNG channel contains a cytoplasmic ligand-binding domain (BD) that includes a ␤-roll (flanked by short helices) and a single C-terminal helix called the C-helix that was previously found to control efficacy (maximal open probability) and selectivity for cGMP versus cAMP. We constructed a series of chimeric CNG channel subunits, each containing a distinct BD sequence (chosen from among six phylogenetically divergent isoforms) fused to an invariant non-BD sequence. We assayed these "BD substitution" chimeras as homomeric CNG channels in Xenopus oocytes to compare their functions and found that the most efficient activation by both cAMP and cGMP derived from the BD of the catfish CNGA4 olfactory modulatory subunit (fCNGA4). We then tested the effects of replacing subregions of the bovine CNGA1 BD with corresponding fCNGA4 sequence and hence identified parts of the fCNGA4 BD producing efficient activation. For instance, replacing either the "hinge" that connects the roll and C-helix subdomains or the BD sequence N-terminal to the hinge greatly enhanced cAMP efficacy. Replacing the "loop-␤8" region (the C-terminal end of the ␤-roll) improved agonist sensitivity for cGMP selectively over cAMP. Our results thus identify multiple BD elements outside the C-helix that control selective ligand interaction and channel gating steps by distinct mechanisms. This suggests that the purine ring of the cyclic nucleotide may interact with both the ␤-roll and the C-helix at different points in the mechanism. Cyclic nucleotide-gated (CNG)1 channels conduct mono-and divalent cations upon activation by the direct binding of the cytoplasmic messengers cAMP and cGMP. These channels are widespread in the nervous system and in a variety of other tissues, and most notably they are essential signaling components in visual and olfactory transduction, where their activation leads both to changes in membrane potential and to the influx of calcium into the cytoplasm (reviewed in Refs. 1 and 2). Functional CNG channels are tetramers (homomeric or heteromeric) of homologous subunits; vertebrates contain a family of six paralogous CNG channel subunit genes in two phylogenetic subfamilies (3), CNGA and CNGB. Distinct combinations of these paralogues are expressed in each tissue type to produce CNG channels whose response parameters are presumably adapted to the physiological requirements of the tissue; these parameters include sensitivity and efficacy (maximal ligandgated open probability) for cAMP and cGMP and selectivity for one agonist over the other. Thus, CNG channels hold promise as targets for tissue-specific pharmacological regulation of signaling through cyclic nucleotide-dependent, electrical, and calcium-dependent pathways. It is therefore important to understand how CNG channel function is determined by the sequences of individual subunits and more generally how structural elements in the channel work t...

Subunit counting in membrane-bound proteins

“…We used the X-fA4 chimera of the bovine CNGA1 and catfish CNGA2 and CNGA4, which had been shown to form functional channels 14 . Although many of the spots bleached in 4 steps, as expected (Fig.…”

Xenopus oocyte prophase I meiotic arrest is released independently from a decrease in cAMP levels or PKA activity

“…For that purpose, we used two membrane channels as sensors for cAMP and its effector PKA: the cyclic nucleotide gated channel (CNG), a cAMP-gated Ca 2+ channel; and the cystic fibrosis transmembrane regulator (CFTR), a PKA-activated Cl − channel (Biel and Michalakis, 2009;Hanrahan et al, 1996). Both channels have been expressed and well characterized in Xenopus oocytes (Bear et al, 1991;Nache et al, 2012;Weber et al, 2001;Young and Krougliak, 2004). A rise in cAMP gates CNG channels, which permeate both mono-and divalent cations, including Ca 2+ .…”

“…This suggests that basal cAMP levels, at least in the sub-plasma membrane domain detected by the CNG channel, were unchanged up to 30 min after P4 treatment. However, the CNG channel clone used is gated by both cGMP and cAMP (Young and Krougliak, 2004), so the lack of response could be because the effect was masked by cGMP in the oocyte.…”

“…We used CNG-expressing oocytes to determine the level of endogenous cAMP in the oocyte and the effectiveness of db-cAMP. Young and Krougliak have previously characterized in Xenopus oocytes the cAMP dose dependence of the recombinant CNG channel, x-fA4, that we used here (Young and Krougliak, 2004). They showed a steep dose dependence of I CNG on cAMP concentrations with a K 1/2 of 1 μM and a saturating current above 200 μM cAMP (see figure 2C in Young and Krougliak, 2004).…”

“…The CNG channel chimeric clone X-fA4 was a gift from Edgar Young (Simon Fraser University, Canada) (Young and Krougliak, 2004). AKAR2 (Lam et al, 2012) was purchased from Addgene and subcloned into the BamHI-EcoRI sites of pSGEM.…”