González-Pérez V , Neely A , Tapia C , González-Gutiérrez G , Contreras G , Orio P , Lagos V , Rojas G , Estévez T , Stack K , Naranjo D .

	Figure 1. Slow inactivation in Shaker IR is weakly sensitive to external TEA. (A) The ionic current traces were elicited by a 15-s pulse to 0 mV protocol schematized at the top of the figure. The TEA concentrations, listed near each trace, were obtained by equivalent replacement of NaCl by TEACl to keep ionic strength constant in the bath solution. The continuous lines on top of each trace are the fits to a single exponential function with time constants of: 4.9, 5.5, 5.8, 5.8, 4.9, and 4.5 s for the traces indicated with [TEA] = 0, 5, 10, 20, 50, and 75 mM, respectively. The dashed line indicates the zero current level. (Inset) Unblocked fractional peak current as a function of the external TEA. Each family of symbol corresponds to a different oocyte. The continuous line was drawn according to an inhibition isotherm with a KD of 18 mM for TEA. (B) Normalized time constant as a function of the external TEA concentration. Each family of symbols corresponds to the same oocytes as in the inset. The continuous line was draw according to the prediction of Scheme 1, with τ/τ0 = (ki×U + kr)−1, where τ/τ0 is the fold increase in time constant, U the fractional unblocked current in the presence of TEA, and ki and kr are equal to 0.19 s−1 and 0.05 s−1, respectively (Oliva et al., 2005). In average for Shaker IR, τ0 = 4.5 ± 0.9 s (n = 12).
	Figure 2. Voltage dependence of Shaker IR channel availability in the presence of external TEA. (A) Ionic currents in the absence of TEA elicited after the protocol schematized at the top of the figure. This protocol consisted of a 60-s prepulse to voltages usually ranging from −70 to −15 mV with 5-mV steps. At the end of this period, channel availability was tested with a pulse to +50 mV. (B) Similar traces in the presence of 40 mM TEACl replacing an equivalent concentration of NaCl. (C) Comparison of the peak current amplitude at the test voltage pulse for the control traces shown in A (open circles) and the 40-mM external TEA traces in B (filled circles). The continuous lines are fits to a Boltzman distribution function with the half inactivation voltages depicted in the figure and an effective valence of z = −6.34 eo for the control traces and z = −6.63 eo for the TEA traces. HP, the holding voltage was −90 mV. Discontinuous lines in A and B indicate zero current level. (Inset) Activation relationship measured at the end of a 50-ms pulse taken before (open circles) and after (filled circles) the inactivation protocols for the experiments shown. Conductances were calculated assuming a reversal potential of −100 mV. Continuous lines are a Boltzman distribution equation with V0.5 = −20.0 and −21.7 mV for the before and after data, respectively, with effective valences of 3.2 and 3.3, respectively.
	Figure 3. Inactivation kinetics in Shaker IR T449S is slowed down by external TEA. (A) Ionic current traces elicited by a 5-s pulse to 0 mV as depicted on the top of the figure. Shown are traces in 0, 1.5, and 3 mM of external TEA added to the recording solution. Continuous lines at the top of the traces are fits to a single exponential function with time constants of 0.38, 0.60, and 0.84 s, for 0, 1.5, and 3 mM TEA, respectively. For longer pulses a slower component becomes evident. (Inset) Unblocked fractional peak current as a function of the external TEA. Each data point corresponds to the average of at least three oocytes. In some instances the error bars are smaller than the symbol size. The continuous line was draw according to an inhibition isotherm with a KD of 4.2 mM for TEA. (B) Normalized time constant as a function of the external TEA concentration. The continuous line is the fit of a linear regression with slope = 0.31 fold/mM, consistent with a KD = 3.2 mM according to Scheme 1. τ0 = 0.35 ± 0.2 (n = 6).
	Figure 4. Voltage dependence of Shaker IR T449S channel availability in the presence of external TEA. (A) Ionic currents in the absence of TEA elicited after the protocol schematized at the top of the figure. This protocol is similar to that shown in Fig. 2, except that the voltage pre-pulses were 30 s long. Inactivation kinetics clearly show two components, with time constants at ∼0.3 and ∼15 s. (B) Similar traces in the presence of 12 mM TEACl added to the recording solution. Consistent with data shown in Fig. 3, TEA not only blocks the current, but also drastically changes its kinetic. (C) Comparison of the peak current amplitude at the test voltage pulse for the control traces shown in A (open circles) and with 12 mM of external TEA in B (filled circles). The continuous lines are fits to a Boltzman distribution function with the half inactivation voltages depicted in the figure using an effective valence of z = −6.56 eo for the control traces and z = −6.92 eo for the TEA traces. Discontinuous lines in A and B indicate zero current level. (Inset) Activation relationship measured 10 ms into the activation voltage pulse taken before (open circles) and after (filled circles) the inactivation protocols for the experiments shown. Conductances were calculated assuming a reversal potential of −100 mV. Continuous lines are a Boltzman distribution equation with V0.5 = −23.4 and −22.5 mV for the before and after data, respectively, with effective valences of 3.2 and 3.4, respectively.
	Figure 5. Diffusional perfusion of internal TEA. (A) COVC current traces elicited by a protocol depicted at the top of the figure. The membrane voltage was held at −90 mV, and voltage was pulsed for 20 ms to +40 mV. Voltage was then returned to −110 mV to induce inward tails of currents. Then, at a different clock rate, a 15-s pulse to 0 mV was applied to assay inactivation kinetics. The variation between traces corresponds to different stages in the diffusional perfusion of internal 5 mM TEACl added on top of the cytosolic solution. Letters a, b, and c indicate traces acquired 3 min before and 3 and 20 min after the beginning of the perfusion of TEA, respectively. For this type of recording, 25 mM Na+ was equivalently replaced by K+ in the external solution to increase the size of the inward tails of currents. (B) The size and kinetics of the tails of currents allow monitoring of the progress of the internal perfusion of TEA and its effect on the deactivation kinetics (see main text for details). The traces shown correspond to an enlargement of the currents recorded at −110 mV in A (left). Single exponential functions were fit to the currents (see Fig. 7). (C) Time course of inhibition by 5 mM of internal TEA of the ionic current elicited by a 0-mV pulse. The vertical dashed line indicates the moment at which we changed the composition of the solution at the internal chamber to initiate the diffusional perfusion of 5 mM TEACl into the cytosolic face. (D and E) Time course of the effect of the internal perfusion of TEA on the time constants of slow inactivation and deactivation (see main text for details), respectively.
	Figure 6. Slow inactivation retardation by internal TEA produces crossing over of the current traces in Shaker IR. Two ionic current traces elicited by a 60-s pulse to −15 mV obtained before (thin line) and 40 min after (thick line) the beginning of the perfusion of internal TEA. The antagonism of TEA on slow inactivation is such that at the end of a 60-s pulse, the total ionic currents in the blocked channels is larger than that in the unblocked one.
	Figure 7. Internal perfusion of TEA delays deactivation and slow inactivation in Shaker IR. (A) Elements of the analysis of the effect of internal TEA on the tails of currents measured at −110 mV. As examples, the first and last traces of the experiment in Fig. 5 are shown. Current deactivations were fit to a single exponential function, and the time constant is shown on top of each trace (discontinuous lines). The amplitudes ao and a are extrapolations of the curves fit to the peak of the preceding capacitive transient, in which a is the amplitude of a given trace and ao is the amplitude of the first one. The ratio a/ao is the unblocked fraction at −110 mV. (B) Plot of the deactivation rate as a function of the unblocked fraction of currents for five different oocytes. Continuous lines are the linear regressions used to estimate the values of α and β (see main text). (C) Elements of the analysis of the effect of TEA on the ionic currents measured at 0 mV. Fitted single exponential functions are shown as discontinuous lines on top of two example traces with the times constants shown, while a and ao have the same meaning as in A. (D) Plot of the slow inactivation rate as a function of the unblocked fraction of currents for the five oocytes shown in B. Identical symbols in B and C correspond to data from the same cell. Continuous lines are linear regressions to estimate the values of TEA-dependent inactivation constant.
	Figure 8. Inactivation kinetics in Shaker IR T449S is not slowed by internal TEA. (A) COVC current traces elicited by a 1-s pulse to 0 mV during the internal diffusional perfusion of 5 mM TEA. (B) Tails of currents are slowed as the ionic current inhibition progresses. (C) Time course of the change on the deactivation and inactivation kinetics. The vertical dashed line indicates the moment at which we changed the composition of the solution at the internal chamber to initiate the diffusional perfusion of 5 mM TEACl into the cytosolic face on the oocyte.
	Figure 9. Voltage dependence of Shaker IR channel availability in the presence of internal TEA. (A) Ionic currents in the absence of TEA elicited using the protocol schematized at the top of Fig. 2 in COVC. (B) Similar traces obtained after 40 min of diffusional perfusion of internal 5 mM TEA. (C) Comparison of the peak current amplitude at the test voltage pulse for the control traces shown in A (open circles) and after the 40-min perfusion of TEA traces in B (filled circles). The continuous lines are fits to a Boltzman distribution function with the half inactivation voltages depicted in the figure using an effective valence of z = −4.6 eo for the control traces and z = −5.81 eo for the traces with TEA. The holding voltage was −90 mV. Discontinuous lines in A and B indicate zero current level.
	Figure 10. Effect of internal and external TEA in the kinetics of recovery from inactivation. (A) Ionic current traces in response to a two-pulse protocol used to measure the rate of recovery from inactivation. From a holding potential of −90 mV, a 15-s conditioning pulse to 0 mV was applied to induce a significant amount of inactivation. Then, after a variable interpulse interval ranging from 10 to 5,000 ms at −90 mV, a second test pulse was applied to measure at the peak the amount of recovery. The top panels show the control traces for TEVC (left) and COVC (right). Note the similarity in the currents obtained using these two modes of recording. The two-pulse recording in the presence of external TEA at 40 mM (left) and after ∼40 min of internal perfusion of 5 mM TEA (right) are shown, rescaled to accentuate the differential effect of TEA on the kinetic component of the recovery (note the different scale bar). (B) Normalized fractional recovery before (open circles) and after (filled circles) the perfusion of external (left) and internal TEA (right). The difference between peak current at the test pulse and the current at the end of the conditioning pulse was normalized according to the relation: 1−[ftest − 1)/(fend − 1)], where ftest and fend are the fractional current at the test (peak) and at end of the conditioning pulse, respectively. The solid lines are fits to a two-exponential function of the form f(t) = 1 + Af × exp(−(t−to)/τf) + As × exp(−(t−to)/τs), where τf and τs are the time constants of both kinetic components of the recovery, −Af and −As are the fractional amplitude of each component, and to is the end of the conditioning pulse. The parameters used for fitting the data on the left panel are: Af = −0.39 ± 0.04, τf = 40 ± 8.4 ms and As = −0.61 ± 0.02, τs = 2,750 ± 274 ms for the control, and Af = −0.58 ± 0.07, τf = 45 ± 13 ms, and As = −0.42 ± 0.05, τs = 1,740 ± 550 ms for the 40 mM external TEA; for the right panel data they are: Af = −0.53 ± 0.04, τf = 29 ± 4 ms and As = −0.44 ± 0.01, τs = 2,800 ± 320 ms for the control, and Af = −0.34 ± 0.05, τf = 34 ± 10 ms and As = −0.67 ± 0.03, τs = 3,500 ± 400 ms for the internally perfused TEA. (Inset) Expanded time scale for the recovery data during the first 100 ms. For this time interval, data were fit to a single exponential function with time constant, τ = 23 ± 3 ms, and amplitude, A = 0.54 ± 0.05 for the control, and τ = 26 ± 13 ms, with A = 0.3 ± 0.03 for data in the presence of TEA. Each data point in the figure corresponds to an average of five to eight different oocytes.
	Figure 11. Effect of internal TEA on the gating currents. (A) Gating currents of Shaker IR channels from a holding potential of −90 mV (top) or 0 mV (bottom), before (left) or 70 min after (right) the beginning of the diffusional perfusion of 5 mM TEA. To be as close as possible to inactivation at equilibrium, the oocyte was kept at a 0-mV holding potential for 5 min before the beginning of each family of voltage pulses. To minimize fast recovery during pulses from 0 mV (see Fig. 10), we kept the pulse duration to 20 ms, a duration balancing the need for a reliable estimation of the base line and recovery. Gating current sizes were stable during the experiments. Although traces at left and right are separated by >70 min, the outward-going gating currents changed in <10%. (B) Outward-going gating charge from either a holding voltage of −90 mV (circles) or 0 mV (squares). The charge measurement made 70 min after the perfusion of TEA (filled symbols) shows a marked disparity to the controls (open symbols) only for the pulses from a holding potential of 0 mV. Q-V relation elicited from a holding potential of −90 mV were fitted to a single Boltzman distribution function with the following parameters: V0.5 = −37.7 mV with z = −2.81 e for the control measurements and V0.5 = −39.0 mV with z = −2.63 e with internal TEA. On the other hand, the Q-V curves elicited from a holding potential of 0 mV were fitted to a two-Boltzman distribution function representing Q1 and Q2 as in Olcese et al. (1997). Parameters used to fit the control charge displacement are: V0.51 = −40.4 mV with z1 =4.27 e having a fractional area of 0.11; and V0.52 = −107 mV with z1 = 1.33 e. Those for the charge displaced in the presence of internal TEA are: V0.51 = −50.8 mV with z1 =3.75 e having a fractional area of 0.59, and V0.52 = −113 mV with z1 =1.2 e.
	Figure 12. The operational voltage dependence of the blockade. (A) Effect of internal TEA on the deactivation versus inactivation. (B) Current inhibition by internal TEA at −110 mV versus the inhibition at 0 mV. See details in the main text. Abscissa data were binned every 0.2 units. The average ± SEM versus the central values of the bin are plotted. The plotted data were clipped at the extreme values; however, >90% of the total data points are represented for each case. In A, this 90% of the data points includes up to 80% of ionic current blockade by internal TEA, whereas in B data include up to a 3.5-fold increase in the deactivation time constant.
	Figure 13. An allosteric kinetic scheme for slow inactivation of Shaker IR. (A) The kinetic scheme devised for U-type inactivation (Klemic et al., 2001) was modified to include blockade by internal TEA. The scheme contains three parallel activation pathways: C-inactivated, normal, and U-inactivated. Internal TEA binds with kon = 2,400 mM−1s−1 and koff = 2,400 s−1. We changed the names of some variables such that: kI = kp, k-I = k-p, kU = ki, and k-U = k-i. All rate constants and allosteric factors are as in Klemic et al. (2001). (B) Simulated time course of macroscopic inactivation in response to a voltage step to 0 mV in the absence (thin trace) and presence of 0.6 mM TEA (thick trace). This TEA concentration is shown because we could not see crossing over of the traces for other TEA concentrations. (C) Normalized voltage dependence of the near equilibrium inactivation in the absence (thin trace) and presence of 5 mM internal TEA (thick trace). TEA produces a shift of −2 mV when fitted to a Boltzman distribution. (D) Normalized recovery from inactivation in the absence and presence of 5 mM TEA. The fast recovery time constant remained ∼15 ms under both experimental conditions, whereas the slow recovery retained a time constant of ∼840 ms. At 0-mV pulses, inactivation time constant changed from 7.7 s in the absence of TEA to 12.9 s with 5 mM TEA. The macroscopic inactivation rate decreased linearly with the fraction of unblocked current (see Fig. 7 D) with a slope of 0.065 s−1 and constant of 0.065 s−1. On the other hand, the macroscopic deactivation rate constant at −110 mV decreased with a slope of 1.5 ms−1 as a function of the unblocked current, consistent with the magnitude and voltage dependence of k-O.

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