Calmodulin preassociates with CNGA2-A4-B1b channels. (a) Ca²⁺ dependence of channel modulation by 100 nM calmodulin. Data are from the same patch and are representative of data from four patches. (b–d) Apocalmodulin can associate with CNGA2-A4-B1b channels without having an effect on cAMP sensitivity. (b) Exposure of channels to 500 nM calmodulin for 30 s in 0.1 μM Ca²⁺ (red trace) or in 10 mM EGTA/0 Ca²⁺ (black trace) does not cause modulation but does induce a Ca²⁺-dependent sensitivity that persists beyond a 3-s calmodulin-free wash (shaded box). Data are from the same patch and are representative of data from 16 patches. Current decline (inset) after calmodulin-free wash periods (shaded box) of 2 s (blue trace) and 3 s (red trace). Data are from the same patch and are representative of data from ten patches. (c) The fraction of channels that are sensitive to Ca²⁺-dependent current decline is a function of the duration of pre-exposure to apocalmodulin. No apocalmodulin exposure (black trace) and 10-s (blue trace) and 1-s (red trace) pre-exposures to apocalmodulin are shown. Data are from the same patch and are representative of data from seven patches. (d) A double point mutation affecting the Ca²⁺-CaM binding site in CNGA2 (F68A V75A; A2_mut) does not perturb preassociation of apocalmodulin to CNGA2-A4-B1b channels or channel modulation by Ca²⁺-CaM. Data are from the same patch and are representative of data from six patches. All records were made at −40 mV. Currents were normalized to the maximal amplitudes, I_max, of each trace. Currents at 7.5 μM cAMP (I_7.5) were used to test CaM effects.

Calmodulin-binding sites in CNGB1b and CNGA4 that mediate Ca²⁺-CaM modulation of CNGA2-A4-B1b channels. (a) Schematic representation of the three rat olfactory CNG channel subunits, showing transmembrane regions S1–S6 (gray) and binding sites for calmodulin (black) and cAMP (crosshatching). Sequence represents the IQ-type apocalmodulin-binding site in the N terminus and 1-8-14-type Ca²⁺-CaM-binding site in the C terminus of CNGB1b. Numbers are signature residues of the 1-8-14-type motif in CNGB1b; asterisk in CaM1 identifies leucine, which was targeted for mutagenesis. (b) Left, CNGA2-A4-B1b channels become insensitive to Ca²⁺-CaM after deletion of the IQ-type CaM1 site of the CNGB1b subunit or an L183E substitution in CaM1. Deletion of the CaM2 site or the N-terminal 74 CNGB1b-specific residues does not compromise calmodulin sensitivity. Right, concentrations for half-maximal channel activation (EC₅₀; mean ± s.d. of five cells) without calmodulin (open bars) and with fully developed Ca²⁺-CaM modulation (black bars) of CNGA2-A4-B1b channels and CNGA2-A4-B1b_mut channels. (c) Schematic representation of the three rat olfactory CNG channel subunits, showing transmembrane regions S1–S6 (gray) and binding sites for calmodulin (black) and cAMP (crosshatching). Sequence represents the IQ-type apocalmodulin binding site in the C-linker of CNGA4; asterisk identifies leucine 292, which was targeted for mutagenesis (d) Left, channel inhibition is lost when CNGA2-A4-B1b channels contain a mutant CNGA4 subunit in which a L292E exchange disables the IQ-like calmodulin-binding site in the C-linker. Right, CNGA2-A4_mut-B1b channels examined as in b. All records were obtained at +40 mV with 50 μM Ca²⁺ and 10 μM cAMP for all subunit compositions. Currents were normalized to the amplitude of the CaM effect.

Ca²⁺-dependent inhibition of native rat olfactory CNG channels. Recordings from inside-out membrane patches excised from dendritic knobs of OSNs. (a) After excision of a patch, almost no current is activated by 7.5 μM cAMP in 50 μM Ca²⁺ (black trace). This Ca²⁺-dependent insensitivity to 7.5 μM cAMP disappeared after 10 min of washing in 10 mM EGTA, and was fully restored by application of 500 nM calmodulin in 50 μM Ca²⁺ (red trace). Holding potential was −40 mV. (b) Dose-response relationship of steady-state CNG current in 50 μM Ca²⁺ obtained from unwashed and washed patches. Mean ± s.d. (three patches). Smooth curves were drawn from the Hill equation, with EC₅₀ values and Hill coefficients of 8.1 μM and 1.8 (washed) and 27.3 μM and 1.9 (unwashed).

Ca²⁺-CaM modulation of CNGA2-A4-B1b channels does not involve the N-terminal Baa motif of CNGA2. (a) Schematic representation of the three rat olfactory CNG channel subunits, showing transmembrane regions S1–S6 (gray) and binding sites for calmodulin (black) and cAMP (crosshatching). Asterisks in the calmodulin-binding site identify amino acids targeted for mutagenesis; numbers are signature residues of the 1-8-14-type Baa Ca²⁺-CaM-binding sites. (b) Left, current decline owing to channel inhibition by 500 nM calmodulin in 50 μM Ca²⁺ (black bar) in inside-out patches of HEK 293 cells expressing (upper) CNGA2-A4-B1b or (lower) CNGA2_mut-A4-B1b channels that lack the 1-8-14-type Ca²⁺-CaM binding site (deletion of residues 61–90). All records were obtained at +40 mV with 50 μM Ca²⁺ and 10 μM cAMP for all subunit compositions, except for those with A2ΔCaM, where 30 μM was used. Currents were normalized to the amplitude of the CaM effect. Right, concentrations for half-maximal channel activation (EC₅₀; mean ± s.d. of five cells) without calmodulin (open bars) and with fully developed Ca²⁺-CaM modulation (black bars) of CNGA2-A4-B1b channels and CNGA2_mut-A4-B1b channels.