Ledwell JL and Aldrich RW (1999), Mutations in the S4 region isolate the final vo...

XB-ART-13451

J Gen Physiol 1999 Mar 01;1133:389-414.

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Mutations in the S4 region isolate the final voltage-dependent cooperative step in potassium channel activation.

Ledwell JL , Aldrich RW .

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Charged residues in the S4 transmembrane segment play a key role in determining the sensitivity of voltage-gated ion channels to changes in voltage across the cell membrane. However, cooperative interactions between subunits also affect the voltage dependence of channel opening, and these interactions can be altered by making substitutions at uncharged residues in the S4 region. We have studied the activation of two mutant Shaker channels that have different S4 amino acid sequences, ILT (V369I, I372L, and S376T) and Shaw S4 (the S4 of Drosophila Shaw substituted into Shaker), and yet have very similar ionic current properties. Both mutations affect cooperativity, making a cooperative transition in the activation pathway rate limiting and shifting it to very positive voltages, but analysis of gating and ionic current recordings reveals that the ILT and Shaw S4 mutant channels have different activation pathways. Analysis of gating currents suggests that the dominant effect of the ILT mutation is to make the final cooperative transition to the open state of the channel rate limiting in an activation pathway that otherwise resembles that of Shaker. The charge movement associated with the final gating transition in ILT activation can be measured as an isolated component of charge movement in the voltage range of channel opening and accounts for 13% ( approximately 1.8 e0) of the total charge moved in the ILT activation pathway. The remainder of the ILT gating charge (87%) moves at negative voltages, where channels do not open, and confirms the presence of Shaker-like conformational changes between closed states in the activation pathway. In contrast to ILT, the activation pathway of Shaw S4 seems to involve a single cooperative charge-moving step between a closed and an open state. We cannot detect any voltage-dependent transitions between closed states for Shaw S4. Restoring basic residues that are missing in Shaw S4 (R1, R2, and K7) rescues charge movement between closed states in the activation pathway, but does not alter the voltage dependence of the rate-limiting transition in activation.

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Species referenced: Xenopus
Genes referenced: tbx2

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	Figure 2. Macroscopic ionic currents and conductance–voltage relations for wild-type Shaker and mutant channels, Shaw S4, and ILT. (Left) Examples of currents from each channel construct recorded from inside-out patches. Currents were elicited by positive voltage steps to the voltages indicated, after a 1-s prepulse to −100 mV from a holding potential of −80 mV. (Right) Normalized conductance plotted as a function of voltage. Normalized conductance–voltage curves were constructed as described in materials and methods. Each symbol represents a different experiment. The smooth curves through the data represent fits to the mean data for each channel with a Boltzmann function, as outlined in materials and methods. The values from these fits are as follows, with n representing the number of experiments used to calculate each mean: Shaker: V1/2 = −40.6 mV, slope factor = 7.2 mV, n = 8; Shaw S4: V1/2 = +80.1 mV, slope factor = +20.4 mV, n = 6; ILT: V1/2 = +73.0 mV, slope factor = 18.3 mV, n = 6. Shaker currents were digitized every 50 μs and filtered at 2 kHz. Currents from ILT and Shaw S4 were digitized every 200 μs and filtered at 2 kHz. The conductance–voltage data for Shaker, Shaw S4, and ILT were taken from Fig. 2 of Smith-Maxwell et al. (1998b).
	Figure 3. Gating kinetics for Shaker, the Shaw S4 chimera, and ILT mutant. (Left) Single exponential fits to the activation time course are shown superimposed on representative current traces for each channel. Note the different time scales. Currents were elicited by positive voltage steps to the voltages indicated, after 1-s prepulses to −100 mV from a holding potential of −80 mV. Current traces were fit with single-exponential functions as described in materials and methods. The time course of Shaw S4 and ILT currents could be fit with a single-exponential function from a beginning current level of between 1 and 5% up to the maximum current level. For Shaker, single exponential fits begin at between 20 and 50% of the maximum current to allow for the large sigmoidal delays characteristic of wild-type Shaker currents. (Right) Time constants of activation and deactivation plotted as a function of voltage. Time constants were obtained from fits of single exponential functions to currents during channel opening and closing (see materials and methods). Data from 10 patches with ILT currents and 14 patches with Shaw S4 currents are shown. ILT currents were digitized every 50 μs and filtered at 8 kHz. Shaw S4 currents were digitized every 200 μs and filtered at 2 kHz, or were digitized every 50 μs and filtered at 8 kHz. The time constants for Shaker and Shaw S4 were taken from Fig. 6 of Smith-Maxwell et al. (1998a) and for ILT were taken from Fig. 4 of Smith-Maxwell et al. (1998b).
	Figure 7. Gating currents in conducting ILT and Shaw S4 channels. (A) Macroscopic ionic currents (top) were elicited by steps to +100 from −140 mV, and then gating currents (bottom) were elicited from the same patches by steps to −120 through 0 mV in 40-mV increments from a prepulse to −140 mV. The ionic currents and the gating currents are displayed using different scales. The results of similar experiments on many patches are summarized in Table I.
	Figure 6. Gating currents from nonconducting Shaw S4 channels. (A) ON gating currents were elicited by steps to +100 to +180 mV in 20-mV increments from a holding voltage of 0 mV. For the purposes of presentation, the data were digitally filtered at 4 kHz. (B) Time constants of Shaw S4 ON gating current decay (open symbols) and ionic current activation (filled circles) as a function of voltage. Time constants of ON gating current decay from four different patches are shown, each symbol representing a different patch. The time constants for ionic current represent the mean values calculated from 14 patches; error bars represent the SD. Time constants were measured as outlined in materials and methods. (C) A semi-logarithmic plot of the Shaw S4 ON gating current decay (open symbols) and ionic current activation time constants (filled circles). Error bars represent SD.
	Figure 4. Gating currents from nonconducting Shaker and ILT channels recorded with cut-open oocyte clamp. (A) Representative Shaker gating current traces. ON gating currents were elicited in response to 50-ms steps to −100, −60, −20, and +20 mV from a 100-ms prepulse of −120 mV. OFF gating currents were elicited by 40-ms steps down to −120 mV after the test pulse. The first 10 ms of the ON and OFF gating current traces are shown. (B) Representative ILT gating current traces. ON gating currents were elicited in response to 50-ms steps to −100, −60, −20, and +20 mV from a 100-ms prepulse to −140 mV. OFF gating currents were elicited by 40-ms steps down to −140 mV after the test pulse. The first 10 ms of the ON and OFF gating current traces are shown. (C) Voltage dependence of charge movement. Normalized QV curves were constructed from the ON gating currents as described in materials and methods. The smooth curves are fits of Boltzmann functions to the mean data as described in materials and methods. The curve through the Shaker data represents a fit with V1/2 = −48.24 mV and the slope factor = 7.1 mV. The curve through the ILT data represents a fit with V1/2 = −86.11 mV and the slope factor = 11.3 mV. (D) Time constants of the decay of the ON gating currents plotted as a function of voltage. The declining phases of the ON gating currents were fitted with single exponential functions. The data were obtained from six experiments for Shaker and four experiments for ILT. Error bars in C and D represent the SEM.
	Figure 5. An additional component of ILT gating charge moves in the voltage range of channel opening. (A) Gating currents from nonconducting ILT channels recorded from inside-out patches. (Left) ON gating currents were elicited in response to 50-ms steps to −140 to +20 mV in increments of 20 mV after a 10-ms prepulse to −140 mV. OFF gating currents were elicited by steps down to −140 mV after the test pulse. The first 10 ms of the ON and OFF gating current traces are shown. (Right) The same patch was then held at 0 mV and ON gating currents at high voltages were elicited by 10-ms steps to +100 to +180 mV in 20-mV increments. (B) Voltage dependence of ON gating charge movement and channel opening. Normalized charge movement (QV, open symbols) and mean conductance (GV, filled circles) for ILT are plotted against voltage. The scale for normalized charge movement is shown on the left and for normalized conductance is shown on the right. ON gating currents were elicited as described in A. Charge measurements from four different patches are shown, each symbol representing a different patch. The conductance data is the mean for six ILT experiments; error bars represent the SEM. The solid line indicates the saturation of charge movement at lower voltages at 0.87. This value was calculated from the average of the normalized charge values at −20 mV for all patches. Charge movement and conductance were calculated as outlined in materials and methods. (C) Time constants of ON gating current decay (open symbols) and ionic current activation (filled symbols) as a function of voltage are shown. Time constants of ON gating current decay from four different patches are shown, each symbol representing a different patch. The time constants for ionic current represent the mean values calculated from seven patches; error bars represent SD. The ON gating currents were elicited as described in A. Time constants were measured as outlined in materials and methods.
	Figure 8. Effects of prepulse voltage on the time course of activation of Shaw S4 and ILT. (A) Macroscopic ionic currents of Shaw S4 and ILT recorded after 100-ms prepulses to either 0 mV (thin lines) or −140 mV (thick lines) from a holding voltage of −80 mV. At the bottom of A, ionic currents of ILT recorded after a 0-mV prepulse and Shaw S4 recorded after a −140-mV prepulse are shown superimposed; to enable comparison across patches with different amounts of ionic current, current traces were scaled to superimpose. Currents were elicited by positive voltage steps to the voltages indicated. (B) The amount of delay at the beginning of the time course of activation for different prepulse voltages plotted over a range of test-pulse voltages. The voltage of the prepulse is indicated beside the name of the channel in the symbol legend. Error bars represent the SEM. Mean values were calculated from seven patches each for Shaw S4 and ILT. The amount of delay was determined from the x intercept of single exponential fits to current traces as outlined in materials and methods.
	Figure 9. Properties of macroscopic ionic currents of Shaw S4: RRK. (A) Representative ionic current traces of Shaw S4:RRK elicited by steps to −40 to +80 mV in 20-mV increments, from a prepulse of −100 mV. (B) Normalized conductance as a function of voltage. Shaw S4:RRK data from 11 patches are shown in open symbols; each symbol represents a different patch. The smooth curves represent fits of a Boltzmann function to mean values of normalized conductance for Shaw S4:RRK, Shaker (left), and ILT (right). Fits to Shaker and ILT are included to facilitate direct visual comparison of the properties of the GV for the different channels. Normalized conductance–voltage curves were constructed as described in materials and methods. The values from these fits are as follows, with n representing the number of experiments used to calculate each mean: Shaker: V1/2 = −40.6 mV, slope factor = 7.2 mV, n = 8; Shaw S4:RRK: V1/2 = +29.9 mV, slope factor = 21.2 mV, n = 6; ILT: V1/2 = +72.9 mV, slope factor = 18.3 mV, n = 6. (C) Time constants of activation and deactivation of Shaw S4:RRK (open symbols) as a function of voltage. Time constants were obtained from fits of single exponential functions to current traces during activation and deactivation, as described in materials and methods. Shaw S4:RRK time constants were measured from nine patches. Single exponential fits (smooth curves) to the voltage dependence of the mean activation data (+10 to +140 mV) and the mean deactivation data (−100 to +10 mV) show equivalent charge values of 0.95 and 0.94 e0, respectively.
	Figure 10. Gating currents of Shaw S4:RRK. (A) Representative gating current traces from cut-open oocyte clamp recordings of nonconducting Shaw S4:RRK. ON gating currents were elicited by steps to −140 to +20 mV in 40-mV increments from a prepulse potential of −180 mV. OFF gating currents were elicited by returning the membrane potential to −180 mV after the test pulse. The first 20 ms of the ON and OFF gating current traces are shown. (B) Voltage dependence of charge movement of Shaw S4:RRK (open symbols). The smooth curves represent fits of a Boltzmann function to mean charge data of Shaw S4:RRK, Shaker (far right), and ILT (middle). The values for the fits are as follows, with n representing the number of experiments used to calculate each mean: Shaw S4:RRK: V1/2 = −106.9 mV, slope factor = 18.3 mV, n = 5; Shaker: V1/2 = −48.24 mV, slope factor = 7.1 mV, n = 6; and ILT: V1/2 = −86.11 mV, slope factor = 11.3 mV, n = 4. (C) Semi-logarithmic plot of time constants of decay of ON gating currents of Shaw S4:RRK (open symbols) and Shaker (filled circles) as a function of voltage. For Shaw S4:RRK, each symbol represents one of five different experiments. For Shaker, the symbols represent mean values calculated from seven experiments; error bars represent SEM. Time constants were determined by fitting the declining phase of the ON gating currents with single exponential functions. Smooth lines represent fits of the time constant data to single exponential functions with equivalent charge values of 0.58 e0 for Shaker and 0.31 e0 for Shaw S4:RRK.
	Figure 11. Relationship between charge movement and activation in Shaw S4:RRK. (A) Voltage dependence of ON gating charge (left axis, open symbols) and normalized conductance (right axis, filled symbols) of Shaw S4:RRK. The threshold at which ionic current can be detected is −30 mV. (B) Effect of prepulse voltage on time course of activation for Shaw S4, ILT, and Shaw S4:RRK. For Shaw S4:RRK, currents were elicited by steps to +100 mV after a prepulse to −40 (thin lines) or −140 (thick lines) mV. For ILT and Shaw S4, currents were elicited by steps to +160 mV after prepulses to 0 (thin lines) or −140 (thick lines) mV. Similar results were seen in five patches of Shaw S4: RRK, seven patches of ILT, and seven patches of Shaw S4.
	Figure 12. (A) Kinetic model for Shaker and ILT. This kinetic model is similar to a Shaker model developed by Zagotta et al. (1994b) except that the last transition to opening has been treated as a separate step. See text for details. (B) A two-state kinetic model for Shaw S4. The 0-mV rate constants and the associated equivalent charge for all rate constants are given in Table II.
	Figure 13. Comparison of observed Shaker and ILT gating currents to model predictions. (A) Comparison of Shaker gating current traces from cut-open oocyte clamp recordings (top) to those predicted by the Shaker model (bottom). ON gating currents were elicited by steps to −100 to +20 mV in 20-mV increments from a prepulse to −120 mV. OFF gating currents were elicited by steps down to −120 mV after the test pulse. For a complete description of the voltage protocols, refer to materials and methods. (B) Comparison of ILT gating current traces from an inside-out patch recording (top) to those predicted in the ILT model (bottom). ON gating currents were elicited with steps to −140 to +20 mV in 20-mV increments from a prepulse to −140 mV. OFF gating currents were elicited by steps down to −140 mV after the test pulse. (C) Voltage dependence of charge movement and (D) voltage dependence of time constants of the decay of the ON gating currents for Shaker (•), ILT (○), with the Shaker and the ILT model predictions shown as solid lines superimposed on the data. Normalized charge–voltage curves were constructed from the ON gating currents as described in materials and methods. Time constants were obtained by fitting the declining phase of the ON gating currents with single exponential functions. The data were obtained from six experiments for Shaker and six experiments for ILT. Error bars represent SEM.
	Figure 14. Comparison of ILT and Shaw S4 ON gating currents in the voltage range of channel opening to those predicted by the 16-state ILT model and the 2-state Shaw S4 model, respectively. (A) ILT ON gating currents in the voltage range of channel opening are shown with traces predicted by the 16-state model (smooth lines) superimposed. For both ILT and the model simulation, ON gating currents were elicited by 10-ms steps to the voltages indicated from a holding voltage of 0 mV. The model traces were scaled to reflect the number of channels in the patch by normalizing to the current level at 1 ms in the +160-mV traces. (B) Shaw S4 ON gating currents in the voltage range of channel opening are shown with traces predicted by the 2-state model (smooth lines) superimposed. For both Shaw S4 and the model simulation, ON gating currents were elicited by 10-ms steps to the voltages indicated from a holding voltage of 0 mV. The model traces were scaled as described in A. The currents in A and B were filtered at 9 kHz during recording, but for this figure, the traces were digitally filtered at 4 kHz to reduce noise and facilitate comparison of data to model-predicted waveforms. (C) Comparison of the ON gating currents in the voltage range of channel opening predicted by the 16-state ILT model (thin lines) and the 2-state Shaw S4 model (thick lines). The current traces were scaled as described in (A). The currents predicted by the two different models are essentially indistinguishable after the first 200 μs.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase