The gating mechanism in cyclic nucleotide-gated ion channels

Abstract

Cyclic nucleotide-gated (CNG) channels mediate transduction in several sensory neurons. These channels use the free energy of CNs' binding to open the pore, a process referred to as gating. CNG channels belong to the superfamily of voltage-gated channels, where the motion of the α-helix S6 controls gating in most of its members. To date, only the open, cGMP-bound, structure of a CNG channel has been determined at atomic resolution, which is inadequate to determine the molecular events underlying gating. By using electrophysiology, site-directed mutagenesis, chemical modification, and Single Molecule Force Spectroscopy, we demonstrate that opening of CNGA1 channels is initiated by the formation of salt bridges between residues in the C-linker and S5 helix. These events trigger conformational changes of the α-helix S5, transmitted to the P-helix and leading to channel opening. Therefore, the superfamily of voltage-gated channels shares a similar molecular architecture but has evolved divergent gating mechanisms.

Figure 1

Inactivation of mutant channels. (a) Mapping CNGA1 residues on the TAX-4 structure (PDB ID: 5H3O): in grey and blue S4-S6, P-loop and C-linker regions of two opposite TAX-4 monomers; in green partial S5 region (S302-V317) of a third monomer. Key residues are in stick representation. Mutant channels that inactivate in a voltage dependent manner (black square); mutant channels that inactivate in a non-voltage dependent manner (red square). (b,c) Current recordings evoked by 1 mM cGMP, during voltage steps at ± 60 mV in symmetrical Na⁺ solution for mutant channels that inactivate in a voltage dependent manner (b) or that inactivate marginally (c). In panel (b) gray traces refer to T355A and F380A, respectively; trace at −60 mV for mutant channel L356A is enlarged in the grey box. (d) Decline of cGMP-activated current for inactivating mutant channels at ± 60 mV. Histograms summarize the average ± s.e.m. and circles indicate data from single experiments at +60 (in red) and −60mV (in black). I _ss/I _max where I _max is the current amplitude immediately after the application of 1 mM cGMP and I _ss is the current at the steady state. (e) The time course of inactivation at +60 and −60 mV obtained by fitting with a single exponential (d). (f) Macroscopic I–V relationships obtained from current recordings as in Figure S2a scaled to the I flowing at +200 mV. Grey scale colors were used for mutant channels that inactivate in a voltage dependent manner and red scale colors for mutant channels that inactivate in a non-voltage dependent manner; R297A (n = 2), T355A ( = 10), L356A (n = 6), E363A (n = 11), T364A (n = 4), F380A (n = 5) and D413A (n = 4). Values represent average ± s.e.m. Unpaired two–tail T-test for T355A, L356A, E363A, T364A, F380A in d: t₆ = −7,911, P < 0.001; t₁₀ = −2,274; P < 0.05; t₈ = 6,881, P < 0.001; t₆ = −5,541; P < 0.01; t₁₀ = 5,652; P < 0.001. Unpaired two–tail T-test for T355A, L356A, E363A, T364A, F380A in e: t₆ = 12.981, P < 0.001; t₆ = −24,02; P < 0.001; t₈ = 19,01, P < 0.001; t₆ = 41,021; P < 0.001; t₁₀ = 14,985; P < 0.001. Further statistical tests for these mutant channels and for all other tested mutants are reported in Supplementary Table 2a,b. ns = not statistically significant (P > 0.05). (n.a.) represents mutant channels with experiments not available, (f.r.) represents mutant channels with no experiments due to fast and complete rundown of the current, (n.e.) represents mutant channels with no experiments because they did not express.

Figure 2

The double mutant channel D413C_R297C can be locked in the open state. (A) Web logo analysis of the sequence surrounding the conserved R297 and D413 residues (marked by an arrow). (B–E) Inactivation and locking in the open state of the double mutant channel D413C_R297C. in symmetrical Na⁺ solution in the presence of 1 mM cGMP at ± 60 mV immediately after the membrane patch excision (B) and after 1 minute (C). The recovery (D) of the current after 1 minute in the closed state. Irreversible lock of the current in the open state (E) after a prolonged exposure to cGMP (10 minutes). (F–G) MTS-2-MTS locking effect in the open state. cGMP-activated currents immediately after the membrane patch excision (F) and after 2 minutes of 100 μM MTS-2-MTS application in the open state (G). (H–I) As in (F) and (G), but in the presence of 100 μM MTS-4-MTS. Red/black lines represent the current evoked by 0/1 mM cGMP. (J) Enlargements of boxes in (H) and (I). (K) Box plot summarize the quantitative noise analysis in the absence (1: n = 6 at +60 mV and n = 6 at −60 mV) and in the presence of 1 mM cGMP, before (2: n = 6 at +60 mV and n = 6 at −60 mV) and after (3: n = 6 at +60 mV and n = 6 at −60 mV) application of MTS-4-MTS. The horizontal line within each box indicates the median of the data; boxes show the twenty-fifth and seventy-fifth percentiles of the data; whiskers show the fifth and ninety-fifth percentiles of the data.

Figure 3

Flexibility in the loops between S4-S5 and S5-P-Helix during CNGA1 channels gating. (A) Superposition of F-D curves from unfolding of CNGA1 in the closed (n = 10) (CNGA1-CS, light and dark red) and open (n = 10) (CNGA1-OS, blue) states. The solid line represents the WLC fitting to the corresponding unfolded peaks, and the numbers represents the approximate peak position in the a.a. chain. Inset represents the ∑∆Lc histogram for the peak at N400 (290 a.a.), implying a conserved unfolding step for both open and closed states. (B) Distribution of unfolded length for the domains S6–S5 for CNGA1-CS (n = 110). (C) As in (B) but for CNGA1-OS (n = 71). (D–E) Cartoon representation of the S5-S6 domains, showing the change in flexibility between the intracellular and extracellular loops of S5 for closed and open state, respectively.

Figure 4

Cysteine scanning mutagenesis shows a state dependent accessibility of S5. (A) Mapping CNGA1 residues on TAX-4 structure (PDB ID: 5H3O) shows the location of key residues in the intracellular portion of S5. (B) as in (A) but for extracellular portion of S5. Key residues are highlighted with red spheres. Different regions (S4–S5 and P-helix) are illustrated in different colours. (C) Comparison between the cGMP-activated currents at ± 60 mV for the mutant channels R297C before (black) and after (grey) 3 minutes application of 2.5 mM MTSET to the intracellular side (MTSET _i) in the closed (c.s.) and open (o.s.) states. (D) Plot representing the comparison of MTSET _i at different concentration (n = 4 for 0.5 mM and n = 5 for 2.5 mM) in the closed state for mutant channels R297C; histograms are the average ± s.e.m. and circles indicate data from single experiments. (E) as in (C) but for the mutant channels Y304C. (F) as in (D) but for the mutant channels Y304C in the open state (n = 4 for 0.5 mM and n = 7 for 2.5 mM). (G,H) as in (E) and (F) but for the mutant channels A322C with the addition of 2.5 mM MTSET to the extracellular side (MTSET _o) and with different concentration of MTSET (n = 3 for 0.5 mM, n = 4 for 1.5 mM and n = 7 for 2.5 mM). (I,J) Histograms representing the average ± s.e.m. for many mutant channels, circles indicate data from single experiments after the application of 2.5 mM MTSET in the open (red) and closed (black) states. The number (n) of experiments for different mutant channels in different states varies between 3 and 8 (see also Supplementary Table 2). (n.e.) represents mutant channels without expression. Significativity is shown only for mutants where the MTSET effect was >30%. Unpaired two–tail T-test in i for R297C and Y304C: t₇ = −7,529, P < 0.001; t₈ = −2,649; P < 0.05. Unpaired two–tail T-test in j for A322C, V348C and L351C: t₈ = −3,668, P < 0.01; t₁₂ = −4,544, P < 0.001; t₆ = −3,109, P < 0.05. Further statistical tests for these mutant channels and for all other tested mutants are reported in Supplementary Table 2c,d. 1-I/Imax represents the normalized current where I is the residual current measured in the presence of 1 mM cGMP after application of MTSET (in different conditions) and Imax is the cGMP-activated current at the beginning of the experiment.

Figure 5

The double mutant channel F317C_Y347C is locked in the open state. (A,B) Mapping CNGA1 residues on TAX-4 structure reveals the presence of hydrophobic interactions. (C) Enlargement of panels a and b illustrating the network of H-bonds and hydrophobic π-interactions. (D) left: current recordings obtained in the absence of cGMP (0 cGMP) for mutant channels F317C_Y347C, elicited by voltage steps from −200 to +200 mV (ΔV = 20 mV); right: after the addition of 1 mM cGMP. (E) left: current recordings obtained in the absence of cGMP; right: 1 minute after the addition of 1 µM dequalinium. (F,G) Current recordings obtained in the absence of cGMP following subtraction of those currents recorded in the presence of dequalinium (F) or after P/-4 procedure for leak and capacitive artefact subtraction (G). (H) Dependence of G/Gmax on V with 1 µM dequalinium and in the absence of cGMP (n = 4, black) and in the presence of 1 mM cGMP (n = 3, red). (I) single channel recordings obtained in the absence of cGMP for mutant channels F317C_Y347C, elicited at different voltages. Amplitude histograms are shown at the right of each trace. isc, single-channel current amplitude. (K) current recordings obtained from the WT CNGA1 channels in the presence of 1 mM cGMP, elicited by the same voltage steps as in (D). (L) Dependence of G/Gmax on V obtained from the recordings of WT CNGA1 channels (K) in the presence (n = 1) of DMA.

Figure 6

Cysteine scanning mutagenesis in S6 and C-linker and S-S bridges between S6 and the P-helix in the open state. (A,B) Web logo analysis of S6 (A) and P-helix (B) in CNG channels based on the alignment given in Supplementary Table S1. (C) Mapping CNGA1 residues on TAX-4 structure (PDB ID: 5H3O) shows the location of mutated residues in S6 and the P-helix. (D) Collected data of the effect of 2.5 mM MTSET_i in the open (red) and closed (black) state in the S6 transmembrane domain and in the C-linker. (E) left: current recordings obtained in the absence (red) and in the presence of 1 mM cGMP (black) at ± 60 mV for mutant channels F380C_L356C. cGMP was added some seconds after setting the voltage command at ± 60 mV. The double mutant does not inactivate and it is gated by cGMP. Right: current recording obtained in the presence of 1 mM cGMP for mutant channels F380C_L356C, elicited by voltage steps from −200 to +200 mV. (F) as in (E) but in the presence of 2 mM DTT present in the patch pipette. (G) Macroscopic I–V relationships of F380C_L356C mutant channels in the presence (n = 3) and in the absence (n = 4) of DTT. The number (n) of experiments for different mutant channels in different states varies between 3 and 8 (for details see also Supplementary Table 2). (n.e.) represents mutant channels without expression, (n.a.) represents mutant channels with no experiments where the expression was too low. (f.r.) represents mutant channels with no experiments where the run-down was too fast. Significativity is shown only for mutants where the MTSET effect was >30%. Unpaired two–tail T-test in d for F380C and V391C: t₁₀ = −7,407, P < 0.001; t₄ = 25,158. Further statistical tests for these mutant channels and for all other tested mutants are reported in Supplementary Table 2e.

Figure 7

Cysteine scanning mutagenesis in S6 and analysis of M-2-M compound in the open and closed states. (A) Mapping of Q417C mutant channel on TAX-4 structure (PDB ID: 5H3O): its distance between adjacent subunits is more than 25 Å apart. (B) left: the cGMP-activated currents at ± 60 mV for the mutant channels Q417C before (black) and after (red) 5 minutes application of 2.5 mM MTSET to the intracellular side (MTSET _i) in the closed (c.s.) and open (o.s.) states. Right: the cGMP-activated currents at ± 60 mV for the mutant channels Q417C before (black) and after (red) 5 minutes application of 100 µM M-2-M in the closed and open states. (C) Histograms representing the average ± s.e.m, after the application of 100 µM M-2-M in the open (red) and closed (black) states. Significativity is shown only for mutants where the M-2-M effect was >30%. Unpaired two–tail T-test for Q409C: t₈ −9,907; P < 0.001. Unpaired two–tail T-test for Q417C: t₁₂ = −6,57, P < 0.05. Further statistical tests for these mutant channels and for all other tested mutants are reported in Supplementary Table 2f. 1-I/Imax represents the normalized current where I is the residual current measured in the presence of 1 mM cGMP after the application of M-2-M and Imax is the cGMP activated current at the beginning of the experiment. (D) Schematic representation of mutant channels Q417C highlighting that residues Q417 belonging to adjacent subunits have a different distance in the closed and open states.

Figure 8

Proposed gating mechanism. (A,B) Cartoon representation of the proposed gating mechanism of CNGA1 from the closed state (A) to the open state (B). Before the binding of CNs, the CNBD is unstructured and D413 and R297 do not interact (A). Upon CNs binding the CNBDs change their conformation and move toward the transmembrane domain (1). The C-linker is then pushed toward the channel core. The interaction between R297 in the intracellular part of S5 transmembrane domain and D413 in the C-linker causes the motion of S5 so that the P-helix can interact with S5 (2). Opening of the gate at the selectivity filter (3). Sequence tracts from S4-S5 to S5 are coloured in blue, S6 in pink, P helices are coloured in magenta. Key residues are highlighted: hydrophobic residues are coloured in red, other residues are grey.