The N terminus of CNGA2 confers PIP₃ sensitivity to CNGA3 channels. (A) Diagrams depicting CNGA2/CNGA3 chimeric subunits are shown on the left (see Methods for splice site locations). Portions of the channel sequence derived from CNGA2 are shown in black; portions derived from CNGA3 are shown in gray. Plotted adjacent is the mean K_1/2 for cGMP before (●) and after (○) 10 μM PIP₃. K_1/2 values ± SD are as follows: A3, 18.6 ± 7 μM before PIP₃ and 20.5 ± 8 μM after PIP₃, three patches; 3322, 24.0 ± 9 μM before PIP₃ and 24.6 ± 11 μM after PIP₃, three patches; A2nA3, 9.3 ± 2.3 μM before and 43.5 ± 13.8 μM after, five patches; 2333, 10.6 ± 4 μM before and 64 ± 19 μM after, four patches; 2233, 12.0 ± 2.4 μM before and 57.3 ± 7 μM after, three patches; CNGA2, 1.8 ± 1 μM before and 23.9 ± 11 μM after, nine patches. (B) Representative cyclic nucleotide dose–response relationships, before (filled symbols) and after (open symbols) application of 10 μM PIP₃, for channels containing A2nA3 subunits. Hill equation parameters (K_1/2, n): (▾), cGMP = 6.8 μM, 2.1 before PIP₃ and (▿) 31.8 μM, 2.4 after PIP₃; (●), cAMP = 1.0 mM, 2.1 before PIP₃ and (○) K_1/2 = 2.1 mM, 2.9 after PIP₃. Data are representative of four different patches.

PIP₃ prevents calmodulin regulation of olfactory CNG channels. (A) CNGA2 cGMP dose–response relationships were measured before (●) and after (▿) application of 250 nM Ca²⁺/CaM. The patch then was washed with 0.5 mM EDTA (■) to remove the CaM. A 10 μM concentration of PIP₃ (◇) caused a right shift that occluded a response to subsequent application of CaM (▴). K_1/2 values: before CaM, 1.7 μM; after CaM, 13.3 μM; EDTA wash, 1.2 μM; after PIP₃, 15.3 μM; PIP₃ + CaM, 13.2 μM. Data are representative of four separate experiments. (B) Representative traces from an inside-out patch containing wtCNGA2/A4/B1b channels stepped to +50 mV and –50 mV and exposed to solutions containing either 500 nM Ca²⁺/CaM or 10 μM PIP₃. Data are representative of three separate experiments. (C) Representative trace from an inside-out patch containing Δ61–90-CNGA2/A4/B1b channels before and after exposure to 10 μM PIP₃. The current activated by 50 μM cGMP was measured at +50 mV every second and normalized to the current activated by 1 mM cGMP. Ca²⁺/CaM (250 nM) was added at 20 s. Data are representative of three separate experiments. (D) After blotting, GST-fusion proteins were probed in an overlay assay by using ≈50 nM FLAG-tagged CaM in 10 μM buffered calcium, either in the presence (below) or in the absence (above) of 100 μM PIP₃. GST-A2N and GST-A2NΔ are as in Fig. 3B; GST-B1bN, proximal N-terminal region (amino acids 677–764) of bovine CNGB1; GST-A4CL, C-linker region (amino acids 271–339) of rat CNGA4. Bound CaM-FLAG was detected by using M2 anti-FLAG antibody. Arrowheads indicate approximate location of molecular mass markers: 49, 38, and 28 kDa. Corresponding blots above and below each other represent identical exposure times. Data are representative of six different experiments.

PIP₃ inhibits CNGA2 channels but not CNGA3 channels. Representative traces from CNGA2 (A) and CNGA3 (B) before and after application of 10 μM PIP₃. Currents were elicited by voltage steps to +50 and –50 mV in the presence of the indicated concentration of cyclic nucleotides. Data are representative of 12 patches for CNGA2 and 6 patches for CNGA3. (C) Two different patches containing CNGA2 channels activated by 1 mM cAMP and held at −50 mV were exposed to 10 μM or 1 μM PIP₃ at 15 s (denoted by arrow). Data were normalized to the current level before PIP₃ exposure. Representative CNGA2 (D) and CNGA3 (E) cyclic nucleotide dose–response relationships before (filled symbols) and after (open symbols) application of 10 μM PIP₃. Hill parameters (K_1/2, n) for CNGA2: (▾) cGMP = 1.6 μM, 2.2 before PIP₃, and (▿) 21.5 μM, 2.1 after PIP₃; (●) cAMP = 51.2 μM, 1.9 before PIP₃, and (○) 1.4 mM, 1.5 after PIP₃; for CNGA3: (▾) cGMP = 21.6 μM, 2.2 before PIP₃, and (▿) 25.8 μM, 2.1 after PIP₃; (●) cAMP = 1.8 mM, 1.3 before PIP₃, and (○) 1.5 mM, 1.7 after PIP₃. Open circles in E overlap and hide the filled circles. Data are representative of three separate experiments each for CNGA2 and CNGA3.

PIP₃ inhibits olfactory CNG channels through a direct interaction with the N terminus of CNGA2. (A) Shown are representative cyclic nucleotide dose–response relationships for Δ61–90-CNGA2, measured before (filled symbols) and after (open symbols) treatment with 10 μM PIP₃. Hill equation parameters (K_1/2, n): (▾) cGMP 22.6 μM, 2.8 before PIP₃ and (▿) 26.9 μM, 2.2 after PIP₃; (●) cAMP = 1.2 mM, 1.4 before PIP₃ and (○) 917 μM, 1.6 after PIP₃. Data are representative of three different experiments. (B) N-terminal regions of CNGA2 and CNGA3 were expressed as GST-fusion proteins and tested for PIP₃ binding in vitro by using PIP₃-agarose beads. Input proteins (Left and Right Lower) and bound proteins (Middle and Right Upper) were identified by immunoblotting with anti-GST antibodies. GST-Grp1PH, positive control pleckstrin homology domain (Echelon); GST-A2N, N-terminal cytoplasmic domain (amino acids 1–138) of rat CNGA2 (AF126808); GST-A2NΔ, amino acids 61–90 deleted; GST-A3N, N-terminal cytoplasmic domain (amino acids 1–164) of human CNGA3 (AF065314); GST-A3NΔ, amino acids 51–108 deleted. Data are representative of four different experiments. (C) Representative cGMP dose–response relationships before (filled symbols) and after (open symbols) application of 10 μM PIP₃ to a patch containing wtCNGA2/A4/B1b channels. Hill equation parameters (K_1/2, n): (▾) cGMP, 2.3 μM, 2.6 before PIP₃ and (▿) 14.6 μM, 1.8 after PIP₃; (●) cAMP, 6.4 μM, 2.8 before PIP₃ and (○) 48.4 μM, 2.0 after PIP₃. (D) Representative cGMP dose–response relationships before and after application of 10 μM PIP₃ to a patch containing ▵61–90-CNGA2/A4/B1b channels. Hill equation parameters (K_1/2, n): (▾) cGMP, 16.3 μM, 2.6 before PIP₃ and (▿) 16.5 μM, 2.7 after PIP_3; (●) cAMP, 70.4 μM, 1.4 before PIP₃ and (○) 51.4 μM, 1.2 after PIP₃. Data are representative of five patches for WT channels and three patches for deletion mutants.