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A constriction formed by the crossing of the second transmembrane domains of ASIC1, residues G432 to G436, forms the narrowest segment of the pore in the crystal structure of chicken ASIC1, presumably in the desensitized state, suggesting that it constitutes the "desensitization gate" and the "selectivity filter." Residues Gly-432 and Asp-433 occlude the pore, preventing the passage of ions from the extracellular side. Here, we examined the role of Asp-433 and Gly-432 in channel kinetics, ion selectivity, conductance, and Ca(2+) block in lamprey ASIC1 that is a channel with little intrinsic desensitization in the pH range of maximal activity, pH 7.0. The results show that the duration of open times depends on residue 433, with Asp supporting the longest openings followed by Glu, Gln, or Asn, whereas other residues keep the channel closed. This is consistent with residue Asp-433 forming the pore's closing gate and the properties of the side chain either stabilizing (hydrophobic amino acids) or destabilizing (Asp) the gate. The data also show residue 432 influencing the duration of openings, but here only Gly and Ala support long openings, whereas all other residues keep channels closed. The negative charge of Asp-433 was not required for block of the open pore by Ca(2+) or for determining ion selectivity and unitary conductance. We conclude that the conserved residue Asp-433 forms the closing gate of the pore and thereby determines the duration of individual openings while desensitization, defined as the permanent closure of all or a fraction of channels by the continual presence of H(+), modulates the on or off position of the closing gate. The latter effect depends on less conserved regions of the channel, such as TM1 and the extracellular domain. The constriction made by Asp-433 and Gly-432 does not select for ions in the open conformation, implying that the closing gate and selectivity filter are separate structural elements in the ion pathway of ASIC1. The results also predict a significantly different conformation of TM2 in the open state that relieves the constriction made by TM2, allowing the passage of ions unimpeded by the side chain of Asp-433.
Figure 1. Relative magnitude of whole cell currents evoked by pH 6.0 of mutants in positions Gly-432 and Asp-433. (A) Normalized currents of Gly-432 substitutions. Columns are the average of â¥12 cells from three different preparations. Error bars are standard deviations. Asterisks indicate significant difference from G432 by unpaired t test, P ⤠0.005. (B) Western blot of lASIC1 detected with anti-FLAG monoclonal antibody from eight oocytes injected with 5 ng cRNA that were biotinylated after measurements of whole cell currents by TEVC. (C) Normalized currents of substitutions of Asp-433. (D) Western blot of oocytes as shown in C, but oocytes expressing N433 and G433 were injected with 50 ng cRNA.
Figure 2. Macroscopic currents of Gly-432 and Asp-433 functional mutants. Representative examples of whole cell currents measured with TEVC. Wild-type channel encodes G432/D433. Substitutions of these positions are indicated for each example. Bars above traces mark the duration of pH 6.5 application. Baseline pH is 7.6. Membrane holding potential is â60 mV. Current and time scales are shown below each trace.
Figure 3. Kinetics of channels with substitutions in residues Asp-433 and Gly-432. (A) Currents from an outside-out patch expressing â¼200 wild-type lASIC1 channels evoked by pH 7.1 and 6.9. (B) Single-channel events of wild-type lASIC1 show long openings of several-second duration and short openings indicated by stippled bars above the current trace. (C) Histogram of duration of open events fitted with three exponentials. (D) Single-channel activity of mutant D433Q. An expansion of the current is shown above the upper trace. (E) Histogram of duration of open events of ASIC1-D433Q. (F) Representative examples of single-channel activity from mutant D433N. (G) Single-channel recordings of D433E. (H) Histogram of duration of open events. (I) Unitary currents of G432C. C, closed state; O, open state. Amplitude and time scales indicated below traces. Holding potential is â60 mV for all examples.
Figure 4. Ca2+ block of lASIC1 with Asp-433 or Gln-433. (A) I-V curves of unitary currents of lASIC1-Asp-433 (filled symbols) and lASIC-Gln-433 (open symbols) examined with various concentrations of Ca2+ in the external solution. All measurements were conducted at pH 7.0 to keep constant the ionization state of Asp-433. Composition of external solution (120 mM NaCl, 2 mM KCl, 15 mM HEPES, and Ca2+) in the indicated concentrations. In 0 mM of solution, Ca2+ was not added to the solution: nominal zero. Each data point is the average of at least 10 independent measurements. Error bars are standard deviation. (B) Affinity of the Ca2+ site measured at various membrane voltages. The lines represent the fit of the data to Eq. 1. Values are KD (0 mV), 3.7 mM and n = 1.3; KD (â20 mV), 3.11 mM and n = 1.2; and KD (â60 mV), 1.78 and n = 1.
Figure 5. Substitution of Asp-433 for Gln maintains the same ion selectivity. (A) I-V curves of lASIC1-D433Q in the presence of external solutions containing 120 mM Na+, Li+, K+, or Cs+ without Ca2+. (B) I-V curves of lASIC1-D433Q in the presence of 120 mM Na+ alone or supplemented with the indicated amounts of Ca2+. Data points are the mean ± SD of five independent measurements. Permeability ratios of monovalent cations were calculated by changes in reversal potential according to Eq. 2. (C) Representative examples of unitary currents of channels bearing Asp-433 or Gln-433 in 0 or 1.5 mM Ca2+ in the activating solution (pH 7.0 recorded at the indicated voltages). Amplitude and time scale are the same for all traces.
Figure 6. Apparent affinity of protons for the activation of mutants G432C and D433Q. Doseâresponse curves of proton activation of mutants G432C and D433Q compared with the parent channel. Lines are fits of the data to Eq. 1. Values of the half-maximal proton concentration for activation or pH50A were 7.2, 7.0, and 6.8, and n = 6, n = 3, and n = 2 for G432C, wild type (WT), and D433Q, respectively.
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