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The acid-sensitive ion channels (ASICs) are a family of voltage-insensitive sodium channels activated by external protons. A previous study proposed that the mechanism underlying activation of ASIC consists of the removal of a Ca2+ ion from the channel pore (Immke and McCleskey, 2003). In this work we have revisited this issue by examining single channel recordings of ASIC1 from toadfish (fASIC1). We demonstrate that increases in the concentration of external protons or decreases in the concentration of external Ca2+ activate fASIC1 by progressively opening more channels and by increasing the rate of channel opening. Both maneuvers produced similar effects in channel kinetics, consistent with the former notion that protons displace a Ca2+ ion from a high-affinity binding site. However, we did not observe any of the predictions expected from the release of an open-channel blocker: decrease in the amplitude of the unitary currents, shortening of the mean open time, or a constant delay for the first opening when the concentration of external Ca2+ was decreased. Together, the results favor changes in allosteric conformations rather than unblocking of the pore as the mechanism gating fASIC1. At high concentrations, Ca2+ has an additional effect that consists of voltage-dependent decrease in the amplitude of unitary currents (EC50 of 10 mM at -60 mV and pH 6.0). This phenomenon is consistent with voltage-dependent block of the pore but it occurs at concentrations much higher than those required for gating.
Figure 1. Kinetic schemes for block (1) and allosteric (2) mechanisms of fASIC. C indicates the closed state with bound Ca2+ or H+. O denotes the open state and D the desensitized state. In Scheme 1 the rate of reaching the O state depends on the rate constants for binding and unbinding of Ca2+, whereas in Scheme 2, the rate depends on the rate constant α.
Figure 2. Effect of divalent cations on the preconditioning solution. Current traces of an outside-out patch from Xenopus oocytes expressing fish ASIC1 activated by a solution of pHo 5.0 containing 1 mM Ca2+. The preconditioning solution, pHo 7.4, contained either 10 or 1 mM Ca2+. Transient inward deflections represent rapidly desensitizing fASIC1 currents. Concentrations of Ca2+ (mM) and pHo are indicated above the currents. Some of the traces have been enlarged to visualize channel activity. Recordings were conducted with 150 mM symmetrical [Na+] and membrane potential of â40 mV. The spikes in the current records are noise induced by the pipette perfusion device.
Figure 3. Activation of fish ASIC1 by external protons. (A) Data are presented as pairs of traces representing outside-out patches activated first (left) by a solution of pHo 5.0 with 1 mM Ca2+. After recovery from desensitization, the same patch was exposed to a test solution of higher pHo (right) as indicated by the bars above the traces. Patches were perfused with preconditioning solution of pHo 7.4 containing 10 mM Ca2+ for 20 s before applying the activating solutions. Experiments were conducted with 150 mM symmetrical [Na+] at a membrane potential of â40 mV. (B) Currents recorded with each of the test solutions were normalized to the corresponding value obtained with pHo 5.0 in the same patch. Normalized currents were plotted against pHo. The curves are predictions from Eqs. 3â5 based on the kinetic analysis (see text). Each point represents the mean ± SD of four to five independent patches.
Figure 4. Proton dependence of apparent rates of activation and desensitization of fASIC. (A) An outside-out patch containing fASIC1 was activated first by a solution of pHo 5.0 and 1 mM Ca2+. The continuous line represents the fit of the currents to Eqs. 4 and 5 with values for rise time, Ïa of â¼1.4 ms, and decay, Ïd of 13.5 ms and peak current of 100 pA. (B) The same patch was subsequently activated by a solution of pHo 7.0 containing 1 mM Ca2+. The trace represents the sum of 12 consecutive such pulses of pHo 7.0. The current is of smaller amplitude, significantly noisier, and has slower time constants, Ïa of 105 ms and Ïd of 15 ms, than with pHo 5.0. (C) Four of the individual responses to pHo 7.0 are shown. (D) Plot of the log of the apparent time constants for activation (Ïa) and desensitization (Ïd) over pHo. Diamonds represent measurements of mean open time of individual channel openings at pHs in the range of 7.2 and 6.8. There is no theoretical meaning of the curves.
Figure 5. Apparent rate constants of activation (αâ²) and desensitization (β) of fASIC as a function of pH. Data of currents obtained at various pHo were fit to Eq. 3 to calculate the valuesof the rates αⲠ(â¯) and β (â¢). Diamonds denote β calculated from the mean open time of channel openings recorded in the pH range of 7.2 to 6.8. The lines are the fitted functions (Eq. 4).
Figure 6. Activation of fASIC1 by decreasing the concentration of external Ca2+. (A) Paired current traces from outside-out patches expressing fASIC1 activated first by pHo 5.0 and 1 mM Ca2+ followed by a second activation by solutions of pHo 7.1 containing decreasing concentrations of Ca2+, from 1 mM to 10 nM, as indicated by the bars above the traces. Patches were preconditioned with pHo 7.4 and 10 mM Ca2+ after exposure to activating solutions. Experiments were conducted with 150 mM symmetrical Na+ and external solutions containing various concentration of Ca2+ that were calculated from a mixture of Ca2+ and EGTA according to the program CAMG, Ver 2 (W.H. Martin) and verified with a Ca2+ electrode. The pipette solution contained 1 mM EGTA and no added Ca2+. (B) Peak currents as a function of [Ca2+] measured at activating pHo of 7.1 and 7.2. Currents were normalized to the value obtained with pHo 5.0 in the same patch. Each point is the mean ± SD of three to five independent patches. The continuous (pH 7.1) and dotted (pH 7.2) lines represent predictions from Eqs. 3â6. To produce the curve at pH 7.2, β was multiplied by 4. (C) Plot of the log of the time constants(s) for activation Ïα (â¯) and desensitization Ïβ (â¢) as a function of pCa. Triangles are estimates of the first latency to channel openings. The straight lines show the fitted function Eq. 6.
Figure 7. Calcium block of fASIC1 channels. I-V plot of unitary current amplitudes of fASIC1 activated by solutions of pHo 6.0 containing 10 nM, 1 mM, or 10 mM Ca2+. Each point represents the average of three or four independent measurements ± SD. Symmetric 150 mM [Na+]. The curves are the simultaneous fits to the equation I=γV/(1+[Ca2+]/Kdeâ(zδVF/RT)), with values for γ of 34 pS, Kd of 74 mM, and δ (fractional electrical distance) of 0.35.
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