Sequence alignment of the S2 domain of the cB3 subunit residues define the <i>Tri-Asp motif</i>.

<p>Asp252, 256 and 262 in canine B3 delineate the highly conserved <i>Tri-Asp motif</i> which is conserved in all CNG channels with relative positions 2 and 3 conserved in the HCN channels. The flanking positions 1 and 3 are acidic residues in the Kv channels aligned here. c = canine; b = bovine; h = human, r = rat; m = mouse. CNGA1 and CNGB1 are rod channels; CNGA2 and CNGA4 are channel subunits expressed in olfactory cilia.</p

Nucleotide-activation of channels comparing wild-type and mutant subunits.

<p>The cAMP efficacy of patches from co-transfected cA3+ hB3-D3/N cells is consistent with these cells expressing homomeric cA3 channels supporting the conclusion that these channels express only the cA3 subunits.</p

Comparison of canine CNG channel properties to human CNG channels.

<p>The K_0.5 values, Hill coefficients (N_h), cAMP efficacy (I_cAMP/I_cGMP), and L-cis Diltiazem block (I_dil/I) of homomeric and heteromeric channels were determined at −60 mV. Averaged calcium to sodium permeability rations were calculated to be 1.52±0.52 (n = 5) for cA3 and 17.7±6.4 for A3+ B3 (n = 4). Comparison permeability ratios for hA3 were not available. Statistical values were SEM except for the two reports indicated with an * which were SD values.</p

<i>Tri-Asp motif</i> in S2 and charged residues of the S1–S4 bundle. A. Topology of the S1–S4 domain of cA3 subunit and localization of the charged residues.

<p>S1 is represented in green, S2 in purple, S3 in yellow and S4 in cyan. Charges not involved in salt bridging are shown in grey, other positive K and R are shown in blue, negative D and E are shown in red. D1, D2 and D3 correspond to D221, D225, and D231 in cA3, respectively. <b>B. Molecular model of a representative S1–S4 domain with all charges of the bundle represented.</b> Color coding is the same as in B. Positive charged side chains charges are in blue, negative charged side chains in red. The <i>Tri-Asp motif</i> residues are noted as D1, D2 and D3 in red. Note that this is a snapshot of the salt bridge network of the P4 subunit voltage sensing domain. Lipids, water, and ions are omitted for clarity. <b>C. Same as B with the addition of water.</b> The Tri-Asp motif residues are depicted as red rods, the solvent accessible volume is delineated by a transparent surface. <b>D. Mapping of the average distance between negative and positive residue pairs.</b> The atom, NZ, is used for K, CZ for R, CG for D, and CD for E. Each subunit is presented separately. The distances were averaged over the last 20 ns of simulation. Distances range from 0 (deep blue) to 40 Å (light green). The charge labels are colored according to the TM segment they belong to. Note that the salt bridge network is rather different from one subunit to the other, highlighting the presence of several conformational changes of similar stability.</p

Structural model of the cA3 homotetramer in a lipid environment. A. Top view:

<p>The four subunits are represented as ribbons. P1 is colored in yellow, P2 in green, P3 in pink and P4 in cyan. Side chains are omitted for clarity. The tetrameric assembly of the two N-terminal helices S5 and S6 forms the central pore. The other TM segments (S1–S4) of each subunit forms a bundle located at the periphery of the pore. <b>B. Top view:</b> The protein is represented as in A. The POPC lipids embedding the channel are represented as Van der Waals spheres, with carbons in white, phosphates in brown, oxygens in red and nitrogens in blue. Water molecules and ions are not shown for clarity. <b>C. Side view with protein and lipids added.</b> The contours of the water-filled volume are represented as a transparent surface. The Na⁺ ions are in orange and the Cl^- in purple. The extracellular domain is located at the top and in the intracellular domain is located at the bottom.</p

Predicted topology of the TM regions of canine and human A3 and B3 subunits.

<p>The TM domains are defined by the crystal structure of the chimeric voltage-gated potassium channel Kv1.2/2.1 (2R9R) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088768#pone.0088768-Long1" target="_blank">[40]</a>. The amino acid sequence identity between the cA3 and hA3 is 82% and is 76% between cB3 and hB3. c = canine, h = human.</p

Cellular localization of YFP-tagged cA3 constructs.

<p>Upper panels: Micrographs illustrating expression of cA3, cA3-D3/N, cA3+ hB3, and cA3+ hB3-D3/N; the arrow indicates a Golgi-like organelle. Intracellular aggregates are seen in all but the cA3-Y cells. Lower panels: averaged expression characteristics constructed from counts of >800 cells. The cA3 and cA3+hB3 expression patterns have a small but significant increase in intracellular aggregates (p = 0.02 on unpaired t-test). Clear, significant differences are seen with expression of mutant D3/N subunits although see text for the interpretation of the cA3+ hB3-D3/N mutant subunit. Scale bar 10 µm.</p

Characterization of CNG channels formed from cA3 alone or co-expressed with hB3.

<p>Recordings from cA3 channels (homomeric) are shown in the left panels and co-expression with hB3 subunits (heteromeric) are shown on the right. Net currents are shown after subtraction of currents measured in the absence of nucleotides. <b>A. Nucleotide-activated currents show cAMP efficacy.</b> Currents were activated with 200 µM cGMP and 5000 µM cAMP at −60 mV (negative currents) and 60 mV (positive currents). The cAMP efficacy is 0.14 for homomeric channels and 0.38 for hetromeric channels. <b>B. IVs with 200 µM cGMP or 5 mM cAMP at ±80 mV.</b> Both the electrode and bath solutions have EDTA and EGTA to eliminate the characteristic Ca²⁺ block of Na⁺ current in CNG channels. In the absence of divalent cations, a slight inward rectification is observed with a 1.6 fold larger current at −60 mV compared to 60 mV for cGMP and 2.2 fold for cAMP in homomeric channels. Little change is seen in heteromeric channels containing the human B3 subunit with rectification of 1.25 fold for cGMP and 1.7 fold for cAMP. <b>C. L-</b><b><i>cis</i></b><b> diltiazem block is greater in presence of hB3 subunits.</b> IVs were recorded with 100 µM cGMP alone or with 25 µM L-<i>cis</i>-diltiazem added to the bath. Net currents were normalized to the maximum cGMP activated current for each patch. <b>D. cGMP and cAMP dose response relationships.</b> Each plot shows the ligand concentration-dependent activation of homomeric or heteromeric channels at −60 mV. Data were taken from a single patch. The K_0.5 for cGMP is 14.2 µM for homomeric and 18.6 µM for heteromeric channels; the N_h is 2.1 for both channel types. The cAMP K_0.5 values are 947 µM and 966 µM, respectively with N_h values of ∼2.5. <b>E. Current reversal potential shifts (E_rev) with 1 mM [Ca²⁺]_i at 200 µM cGMP.</b> Each panel shows net currents activated by 200 µM cGMP with and without 1 mM [Ca²⁺]_i. Voltage ramps were −20 mV to 20 mV. The E_rev shift of homomeric channels is −0.47, whereas that of the heteromeric is −4.02.</p

Complex cellular phenotype of V644del mutant channel.

<p><b>(A)</b> Cellular localization of YFP-tagged wild-type canine CNGA3 and CNGA3-V644del mutant in HEK tsA201 cells. Cells transfected with the wild-type construct showed specific fluorescence pattern of expression limited to the plasma membrane and Golgi-like organelles (arrow); an evident increase in intracellular aggregates was observed in cells transfected with V644del mutant construct consistent with abnormal trafficking and potential ER retention. Scale bar: 10μm. <b>(B)</b> cGMP- and cAMP-activated currents recorded from CNGA3-WT and a responsive patch expressing V644del mutant channels. Approximately 40% of V644del mutant patches had no cGMP-activated currents, in contrast to 100% responsive patches from the CNGA3-WT-transfected cells. This partial loss of channel activity might reflect incomplete subunit assembly associated with disruption of the coiled-coil structure as depicted in the simulation studies. The responsive patches showed cyclic nucleotide-activated currents with similar characteristics to WT channels. <b>(C)</b> Histograms of subcellular localization patterns monitored in HEK tsA201 cells co-transfected with V644del and CNGA3-WT cDNA constructs. Cells were transfected with either CNGA3-WT or CNGA3-V644del or both constructs at the indicated ratios. Each cell count represents >300 cells from at least 2 transfections (mean% ± SD).</p

The V644 deletion alters CLZ core structure indicating unfolding of coiled-coil complex.

<p><b>(A)</b> Sequence alignment of the CLZ (C-terminal leucine zipper) domains in CNGA-type subunits and conservation of the V644 residue. Note that canine V644 corresponds to V630 of human CNGA3. c = canine; h = human. <b>(B)</b> Helical wheel diagrams looking down the superhelical axis of the CLZ coiled-coil from the N- to C- terminus. Heptad <i>a</i> positions are marked in pink, <i>d</i> positions are denoted in magenta, and V630 is shown in red. The V630del shifts residues from <i>b</i> and <i>e</i> heptad positions to the core <i>a</i> and <i>d</i> positions and causes the predominantly hydrophobic residues of the coiled-coil core in the wild-type structure (left) to shift destabilizing charged and small residues in the mutant complex (right). Sequence diagram of the wild-type and V630del mutant heptad is shown below. <b>(C)</b> Molecular dynamics trajectories and models of the wild-type and mutant CLZ trimeric structures. Snapshots before (t = 0 ns) and after (t = 325 ns) simulation for the wild-type CNGA3-CLZ (left). The CLZ core clearly remains intact with small perturbations near the solvent exposed N- and C- termini over long timescales. Snapshots before (t = 0 ns) and after (t = 116 ns) simulation for the V630del mutant model (right). The V630 deletion dramatically alters the previously stabilizing residue-residue interactions, leading to disruption of the CLZ coiled-coil and helical structure. Small red and blue spheres correspond to oxygen and nitrogen atoms, respectively. See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138943#pone.0138943.s004" target="_blank">S4</a></b>and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138943#pone.0138943.s005" target="_blank">S5</a> Figs</b> for the full-length molecular dynamics simulations.</p