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. 2014 Jan 7;106(1):134-44.
doi: 10.1016/j.bpj.2013.11.025.

Mutations in the S6 gate isolate a late step in the activation pathway and reduce 4-AP sensitivity in shaker K(v) channel

Evelyn Martinez-Morales  1 , Dirk J Snyders  1 , Alain J Labro  2
Affiliations

Mutations in the S6 gate isolate a late step in the activation pathway and reduce 4-AP sensitivity in shaker K(v) channel

Evelyn Martinez-Morales et al. Biophys J. .

Abstract

Kv channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10-20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker's sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a α-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.

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Figures

Figure 1
Figure 1
Sequence alignment of Shaker Kv channels with a simplified gating model. (A) The sequence alignment of S6c from different Kv families reveals a highly conserved proline-x-proline motif (PxP), where x is a hydrophobic residue which in Shaker is a valine. It has been proposed that this motif confers flexibility to S6c and forms a hinge region of the BC gate (25). The residues (V474 and V478) that according to a MD simulation (using KcsA as template) are involved in 4-AP binding are highlighted in gray. Interestingly, the Kv7 channels do not possess a full PxP motif and are as KcsA insensitive to 4-AP inhibition. (B) A simplified transition state diagram for the Shaker Kv channel. The different VSD transition states are represented on top with below the corresponding state of the BC gate. Each of the four VSDs can move largely independent from the Down state up to the Activated state. When the VSDs are in their down or activated state the channel is not conducting and the BC gate resides in the closed and activated-not-open (Activated-NO) state, respectively. When all four VSDs have reached their activated state, they transition in a subunit-cooperative way to their Up state, which corresponds with the fully open state of the BC gate. Gating currents provide information on the VSD movements and show that 80–90% of the total gating charges move during their independent reorientation(s) from the down to activated state. The remaining 10–20% of the gating charge is associated with the cooperative transition(s) from Activated to Up, which is accompanied by BC gate opening.
Figure 2
Figure 2
Ionic current measurements of Shaker-IR and P475A. (A) On top are the pulse protocols and below are the corresponding current recordings obtained for Shaker-IR control. The left protocol was used to determine current activation (Iac) and the right protocol for characterizing current deactivation (Ideac). The horizontal bar at the start of the recordings indicates, here and in all other represented ionic current recordings, the zero current level. (B) Representative Iac and Ideac recordings for the mutant P475A are displayed on the left and right, respectively. Both Iac and Ideac were strongly slowed down in P475A compared to control and stronger depolarization were required to reach saturation of the tail currents obtained at −20 mV (note the difference in scale bars and pulse protocols with those of panel A). (C) Panel displays representative current recordings from mock-transfected HEK293 cells (cells were only transfected with the GFP marker). (D) Voltage-dependence of BC gate opening: GV curves for both Shaker-IR control (white circles) and P475A (black circles) were determined from the normalized tail current amplitudes obtained with an activation protocol as shown in panels A and B. Solid lines represent the average fit with a Boltzmann distribution. (E) Displayed on top is an Iac recording of P475A elicited at +100 mV and approximated with a single exponential function. Below the same Iac recording approximated with a double exponential function yielding a clearly better fit with a faster (dark gray) and slower (light gray) component. Both insets show a scaled up view of the first 200 ms of Iac fitted with a single or double exponential function. (F) Time constants of BC gate opening and closure obtained by fitting Iac and Ideac with a single or double (for P475A Iac) exponential function (note the semilogarithmic axis).
Figure 3
Figure 3
Gating current measurements of Shaker-IR-W434F and P475A. (A) Gating current recordings of Shaker-IR-W434F control obtained with activation (left) or a deactivation (right) protocol. On the left, a scaled up view of the activating gating currents (IQac) at +20 mV (blue) and +60 mV (red) are shown as inset. With increasing depolarization strengths the IQac kinetics accelerate causing the IQac tracings to cross each other. Inset on the right shows a scaled up view of the deactivating gating currents (IQdeac) at −90 mV (blue) and −120 mV (red). Similar to IQac, IQdeac decay accelerated with stronger repolarizations. (B) Representative IQac (left) and IQdeac (right) recordings of P475A. Insets show a scaled up view of both IQac and IQdeac obtained with the activation protocol. Similar to control (panel A), IQac accelerated with stronger depolarizations, which is highlighted by coloring the recordings at 0 mV (green), +50 mV (blue), and +130 mV (red). Scale up view of the IQdeac recordings at −90 mV shows that IQdeac did not slow down up to prepulse depolarizations of +50 mV in strength (note the overlap of the green and blue recording, which were obtained upon a 0 and +50 mV prepulse, respectively). However, upon a +130 mV prepulse (red trace) there was a reduction in IQdeac amplitude suggesting that the IQdeac kinetics were slowed down (larger scale of this scaled up view is shown in the Supporting Material). Right panel displays IQdeac recordings that were elicited with a deactivation pulse protocol by stepping after a 150-ms +90 mV prepulse to potentials between 0 and −120 mV. The blue and the red trace were obtained at −80 mV and −120 mV, respectively. (C) QV curves (symbols and line, which represent average fit with a single or sum of two Boltzmann distributions) of Shaker-IR-W434F (black) and P475A (blue) obtained by integrating and normalizing the IQac recordings. For comparison, the GV curves are represented in dotted lines. Note, the QV curve of P475A displayed two gating charge components whereby the first component matched the QV curve of Shaker-IR-W434F, whereas the second component was shifted by ∼ +95 mV and corresponded to the shift in the GV curve. (D) Voltage-dependency of the IQac (circles) and IQdeac (triangles) time constants ± SE for Shaker-IR-W434F (gray symbols, n = 8) and P475A (blue symbols, n = 6). (E) Envelope pulse protocol to determine in P475A the effect of prolonging the prepulse depolarization on the speed of IQdeac decay (τ_IQdeac), which was elicited by repolarizing to −100 mV. With longer depolarization times at +100 mV there was a gradual decrease in IQdeac amplitude, most likely because of a slowing down in τ_IQdeac. Approximating the decay in IQdeac amplitude with a single exponential function (blue dotted line) yielded a time constant of 58.8 ms in the represented recording.
Figure 4
Figure 4
Effect of 4-AP on the ionic currents of Shaker-IR and P475A. (A) Concentration-dependent inhibition of the ionic currents of Shaker-IR; shown for a +20 mV depolarization in presence of different 4-AP concentrations. (B) Concentration-effect curve of 4-AP for Shaker-IR obtained by plotting the normalized Iac currents from pulse protocols shown in panel A as a function of 4-AP concentration; the solid line represents the fit with the Hill equation. (C) Ionic currents of P475A elicited using the pulse protocol shown on top. Represented recordings were the steady-state currents after repetitive pulsing to +90 mV in control condition, in presence of 1 mM and 10 mM 4-AP. In contrast to Shaker-IR control (panel A), there was no inhibition of the current amplitude even with a 4-AP concentration of 10 mM. (D) Current density of P475A (obtained by dividing the current amplitude by the cell capacitance) in control condition (black circles), in presence of 1 mM (triangles), and 10 mM (squares) 4-AP, and upon subsequent wash-out of 4-AP (white circles) (n = 4). To see this figure in color, go online.
Figure 5
Figure 5
Effect of 4-AP on the gating currents of Shaker-IR-W434F and P475A. (A) IQac and IQdeac current of Shaker-IR-W434F in control condition (black) and presence of 1 mM 4-AP (red) elicited with a depolarization to +20 mV and subsequent repolarization to −90 mV. Note that IQdeac decayed markedly faster in the presence of 4-AP. (B) To determine whether 4-AP prevented the slowing of τ_IQdeac in P475A, we used a similar envelope pulse protocol (represented on top) as in Fig. 3E. The gating current recordings obtained in the presence of 10 mM 4-AP clearly showed a reduction in IQdeac amplitude (indicative of a slowing in τ_IQdeac) upon prolonged duration at +100 mV. This slowing in τ_IQdeac developed in the represented recording with a time constant of 125 ms, which was similar to control condition (absence of 4-AP, Fig. 3E). (C) QV curve (obtained from integrating IQac) of Shaker-IR-W434F in control conditions (white circles) and in presence of 1 mM 4-AP (black circles). Normalizing the total charge movement in presence of 4-AP to the charge movement in control conditions showed a 15 ± 5% (n = 3) loss in gating charge. In contrast, P475A displayed a similar QV curve with two gating charge components in control conditions (blue circles) and in presence of up to 10 mM 4-AP (red circles). Fitting the QV curves with a sum of two Boltzmann distributions indicated that V1/2 and slope factor values were not changed significantly after 4-AP application (Table 1). Note, that the amplitude of the second gating charge component of P475A matched the loss in gating charge observed in Shaker-IR-W434F upon 4-AP application. (D) τ_IQac (circles) and τ_IQdeac (triangles) ± SE for Shaker-IR-W434F (n = 3) in control conditions (white) and in presence of 1 mM 4-AP (black). τ_IQac (circles) and τ_IQdeac (triangles) kinetics of P475A in control conditions and in presence of 10 mM 4-AP are represented in blue and red, respectively (n = 5). In contrast to Shaker-IR-W434F, both the τ_IQac and τ_IQdeac kinetics were not affected by 4-AP in P475A. Note that the τ_IQdeac kinetics were markedly accelerated in Shaker-IR-W434F after 4-AP application and that these faster τ_IQdeac values (black) matched the τ_IQdeac kinetics of P475A.
Figure 6
Figure 6
Quinidine block of Shaker-IR and P475A. (A) Concentration-dependent inhibition of ionic currents of Shaker-IR by quinidine. Represented traces are the steady-state currents at 0 mV depolarization in presence of different quinidine concentrations. (B) Representative ionic current inhibition for P475A by different concentrations of quinidine. (C) Concentration-effect curve of Shaker-IR (open circles) and P475A (black circles) for quinidine obtained by plotting the normalized Iac currents from pulse protocols shown in panels A and B as a function of quinidine concentration. The solid line represents the fit with the Hill equation. To see this figure in color, go online.
Figure 7
Figure 7
Biophysical properties of the P475G mutation. (A) Representative Iac and Ideac recordings for the mutant P475G are displayed on the left and right, respectively. (B) Representative IQac recording of P475G using the activation pulse protocol shown on top. (C) Voltage-dependence of BC gate opening (GV curve) and VSD movement (QV curve) for P475G. The GV curve of P475G (blue circles, n = 5) was shifted by ∼ +45 mV toward more positive potentials compared to Shaker-IR control (black dotted line). The blue line represents the average fit with a single Boltzmann equation. The QV curve of P475G (red circles, n = 4) was obtained by integrating and normalizing the IQac recordings obtained with activation protocol shown in panel B. Note, P475G displayed, similar to P475A, a QV curve with two gating charge components whereby the first component carried ∼78% of the gating charge and superposed on the QV curve of Shaker-IR-W434F control (black). The second component carried the remaining 22% of the gating charge and matched the GV curve (blue line). (D) Voltage-dependent time constants of BC gate opening and closure in P475G (blue) obtained by fitting Iac with a double and Ideac with a single exponential function. The τ_Iac (even the fast one) and τ_Ideac kinetics were clearly slower than those of Shaker-IR control (gray). Voltage-dependency of τ_IQac ± SE for P475G (red, n = 8) and Shaker-IR-W434F (black). (E) Increasing gradually the conditioning prepulse duration at +40 mV (pulse protocol is shown on top) resulted in P475G in a gradual decrease in IQdeac amplitude, which reflects a slowing down of τ_IQdeac. Approaching the gradual decrease in IQdeac with a single exponential (blue dotted line) yielded a time constant of 6.5 ms. (F) Ionic currents of P475G after pulsing to +80 mV (pulse protocol shown on top) in control condition (black), and in presence of 1 mM (red), 3 mM (green), and 10 mM (blue) 4-AP. (G) Current density of P475G in control condition (black), in presence of 1 mM (red), 3 mM (green), and 10 mM (blue) 4-AP (n = 4). Over the voltage range tested, no difference in the current density was observed upon 4-AP application.
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References

    1. MacKinnon R. Determination of the subunit stoichiometry of a voltage-activated potassium channel. Nature. 1991;350:232–235. - PubMed
    1. Labro A.J., Snyders D.J. Being flexible: the voltage-controllable activation gate of kv channels. Front Pharmacol. 2012;3:168. - PMC - PubMed
    1. Blunck R., Batulan Z. Mechanism of electromechanical coupling in voltage-gated potassium channels. Front Pharmacol. 2012;3:166. - PMC - PubMed
    1. Vardanyan V., Pongs O. Coupling of voltage-sensors to the channel pore: a comparative view. Front Pharmacol. 2012;3:145. - PMC - PubMed
    1. Long S.B., Campbell E.B., Mackinnon R. Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science. 2005;309:897–903. - PubMed

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