Exploration of the pore structure of a peptide-gated Na+ channel

Abstract

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.

Fig. 1.(A) Sequence alignments for TM1 and TM2 regions within different members of the ENaC/Degenerin gene family. The alignment was obtained with the GCG Pileup program. Identical and similar residues are printed white on black and white on gray, respectively. Acid-sensing ion channels (ASICs), brain–liver–intestine amiloride-sensitive Na⁺channel (BLINaC) and epithelial Na⁺channel (ENaC) correspond to the rat sequences; MEC4 and UNC105 are C.elegansdegenerins and dGNaC1 is a Drosophilagonad-specific amiloride-sensitive Na⁺channel. Note the low conservation between the sequences of TM1 when compared with TM2. (B) Current–voltage relationship for wild-type FaNaC expressed in HEK293 cells, in the presence of 30 µM FMRF-amide. Cells were maintained in the whole-cell configuration and voltage steps were applied from –100 to +100 mV in 20 mV increments. (Circles) Symmetrical conditions (140 mM NaCl, 0 mM KCl pH 7.4) between intracellular and extracellular medium. (Squares) Extracellular medium: 95 mM KCl, 35 mM NaCl; intracellular medium: 140 mM NaCl, 0 mM KCl. (Diamonds) Residual currents in the absence of FMRF-amide. Similar residual currents were observed in the presence of 30 µM FMRF-amide and 100 µM amiloride.

Fig. 2.Susceptibility of wild-type FaNaC and several mutants to 1 mM MTSET. Positive transfectants in the whole-cell clamp configuration were maintained at –40 mV. Bath and pipette solutions were of identical composition (140 mM NaCl, 10 mM HEPES, 1 mM CaCl₂pH 7.4). Cells were briefly perfused twice with the same solution containing 30 µM FMRF-amide to determine the maximal amplitude of the stimulation (I_max) before being rinsed until the baseline current was reached, and then perfused with 1 mM MTSET in the same solution for 3 min. The third peak corresponds to the channel stimulation by 30 µM FMRF-amide after 3 min of MTSET application followed by 1 min of rinse. In these conditions, the wild-type channel (WT) and mutants such as Ile75Cys are not affected by 1 mM MTSET, while mutants such as Val74Cys show a marked inhibition. Note that the Ser86Cys mutant, which exhibits a strong desensitization upon FMRF-amide repeated stimulations, regains activity and does not desensitize following 3 min of 1 mM MTSET exposure. F, 30 µM FMRF-amide; R, rinse.

Fig. 3.Histogram summarizing the sensitivities of the cysteine mutants of the FaNaC first transmembrane segment following 3 min of 1 mM MTSET application, and the corresponding SEMs. Experimental procedures were carried out as described in Figure 2and in Materials and methods. Data for each mutant were obtained from at least six independent recordings and the percentages of inhibition calculated as (I_max– I)/I_max, where I_maxis the current amplitude before 3 min of MTS application, and Ithe current amplitude following MTS application. Cysteine mutants at positions 74, 76, 79, 83, 85, 86, 89 and 90 are strongly affected by MTSET (Student’s t-test, p<0.001), and the corresponding histogram bars are represented in dark grey.

Fig. 4.(A) Open channel measurements for the Leu72Cys mutant. Positive transfectants in the whole-cell clamp configuration were maintained at –40 mV with the same bath and pipette solution (140 mM NaCl, 10 mM HEPES, 1 mM CaCl₂pH 7.4). Following one rapid stimulation with 30 µM FMRF-amide, the clamped cells were perfused for 3 min with 30 µM FMRF-amide plus or minus 1 mM MTSET. Note the stability of the FMRF-amide-evoked current, and its exponential decay in the presence of MTSET. F, 30 µM FMRF; R, rinse; M, 1 mM MTSET. The same protocol has been applied for all the mutants that were shown to be accessible to MTSET in the closed configuration, and the recordings are similar, apart from mutants Ala71Cys, Leu85Cys and Ser86Cys, which inactivated upon continuous FMRF-amide stimulation. (B) Histogram summarizing the sensitivity of the mutants to 1 mM MTSET in the presence and absence of FMRF-amide. Experimental procedures were carried out as described in (A), and the results were compiled from at least six independent experiments, as described in the legend to Figure 3 and in Materials and methods. Note that the percentages of inhibition are similar in the open and closed configuration, apart from position 72, which becomes unmasked upon channel opening.

Fig. 5.Mapping of the charge selectivity filter and amiloride blockage positions. This histogram shows the accessibility of the MTSET-sensitive mutants to the anionic 10 mM MTSES in the closed configuration and the protection from 1 mM MTSET inhibition by 100 µM amiloride. For MTSES accessibility, the experimental procedure is the same as in Figure 2, with 10 mM MTSES replacing 1 mM MTSET. For amiloride protection, clamped cells were tested for FMRF-amide activation, perfused with a 100 µM amiloride solution for 3 min, rinsed, and the response to FMRF-amide was re-tested. This allowed us to verify that amiloride could be rinsed efficiently in the closed configuration. The amiloride solution was then switched after 30 s to 100 µM amiloride plus 1 mM MTSET for 3 min. The cells were then rinsed and tested for residual FMRF-amide activation. For comparison, 1 mM MTSET inhibition of the various mutants in the closed configuration is shown on the same graph.

Fig. 6.Effect of MTSET on wild-type and mutated ASICs. Oocytes injected with ASIC1a, ASIC1b and ASIC2a were exposed to MTSET (2.5 mM) for 5 min, and the percentages of inhibition of the H⁺-elicited currents were determined as described in Materials and methods. The ASIC1a and 1b isoforms possessing cysteines at positions corresponding to pore-facing residues of FaNaC (see inset) are significantly affected by the sulfhydryl reagent, whilst the ASIC2a isoform, which does not possess any cysteine in its first transmembrane segment is insensitive to MTSET. Introducing a cysteine at position 58 converts ASIC2a into a MTSET-sensitive isoform. However, the cysteine mutant introduced at position 49 (equivalent of position 74 in FaNaC) is not affected by MTSET. The number of oocytes tested in each condition is given above the histograms (mean ± SEM).

Fig. 7.Schematic representation of the ion pore of FaNaC deduced from the substituted cysteine accessibility method experiments. For simplicity, only one subunit is represented, and TM2 is symbolized by a cylinder.