Haddad GA and Blunck R (2011), Mode shift of the voltage sensors in Shaker K+ ...

XB-ART-43800

J Gen Physiol 2011 May 01;1375:455-72. doi: 10.1085/jgp.201010573.

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Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain.

Haddad GA, Blunck R.

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The voltage sensors of voltage-gated ion channels undergo a conformational change upon depolarization of the membrane that leads to pore opening. This conformational change can be measured as gating currents and is thought to be transferred to the pore domain via an annealing of the covalent link between voltage sensor and pore (S4-S5 linker) and the C terminus of the pore domain (S6). Upon prolonged depolarizations, the voltage dependence of the charge movement shifts to more hyperpolarized potentials. This mode shift had been linked to C-type inactivation but has recently been suggested to be caused by a relaxation of the voltage sensor itself. In this study, we identified two ShakerIR mutations in the S4-S5 linker (I384N) and S6 (F484G) that, when mutated, completely uncouple voltage sensor movement from pore opening. Using these mutants, we show that the pore transfers energy onto the voltage sensor and that uncoupling the pore from the voltage sensor leads the voltage sensors to be activated at more negative potentials. This uncoupling also eliminates the mode shift occurring during prolonged depolarizations, indicating that the pore influences entry into the mode shift. Using voltage-clamp fluorometry, we identified that the slow conformational change of the S4 previously correlated with the mode shift disappears when uncoupling the pore. The effects can be explained by a mechanical load that is imposed upon the voltage sensors by the pore domain and allosterically modulates its conformation. Mode shift is caused by the stabilization of the open state but leads to a conformational change in the voltage sensor.

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Species referenced: Xenopus laevis
Genes referenced: kcna2 tbx2

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	Figure 1. Uncoupling mutants I384N and F484G. (A) Location of the uncoupled mutants in the Kv1.2 crystal structure (Long et al., 2005a). Shown is a single subunit. I384 is located on the S4-S5 linker, and F481 and F484 are located on the C-terminal S6, which anneals to the S4-S5 linker. (right) Magnification of the interaction site. (B) Gating currents of ShakerIR-W434F, ShakerIR-W434F-I384N, and ShakerIR-W434F-F484G in response to a series of depolarizing pulses from a holding potential of Vhold = −90 mV to a potential of −120 to 60 mV in steps of 10 mV for 50 ms. (C) QVON and QVOFF of ShakerIR-W434F, ShakerIR-W434F-I384N, and ShakerIR-W434F-F484G. (D) Fluorescence voltage (FV) and conductance voltage (GV) relations for ShakerIR-A359C, ShakerIR-A359C-I384N, and ShakerIR-A359C-F484G elicited from a series of depolarizing pulses from a holding potential of Vhold = −90 mV to a potential of −120 to 60 mV/200 mV in steps of 10 mV for a duration of 50 ms. QV and GV are far separated in the uncoupled mutants I384N and F484G. (C and D) Data are shown as mean ± SD.
	Figure 2. Mode shift is removed in uncoupled mutants I384N and F484G. (A) Gating and ionic currents in response to a series of depolarizing pulses from a holding potential of Vhold = −90 mV to a potential of −120 to 180 mV in steps of 10 mV elicited from ShakerIR-I384N. Both gating and ionic currents are detectable. (B) Time constants from exponential fits of the gating currents (saturating depolarizations) obtained from W434F (n = 7), W434F-I384N (n = 6), and W434F-F484G (n = 4). (C) QV curves were elicited for holding potentials at −90 (QV90) and at 0 mV (QV0) of ShakerIR-W434F, ShakerIR-W434F-I384N, and ShakerIR-W434F-F484G in response to a series of depolarizing pulses from −160 to 60 mV in steps of 10 mV. No shift of the QVs is observed in the uncoupled mutants (P > 0.3) except for W434F (P < 0.0001). (D) QV relations of ShakerIR-W434F in response to a series of pulses from a given holding potential (HP) to variable voltages. (right) V1/2 of single Boltzmann fits as a function of the holding potential of the data on left. The shift occurs at voltages between −60 and −40 mV. (B–D) Data are shown as mean ± SD.
	Figure 3. Open state stabilization, strong and weak coupling. (A) Gating currents of ShakerIR-W434F-Y485A in response to a series of depolarizing pulses (50 ms) from a holding potential (HP) of −90 mV to voltages between −120 and 60 mV in steps of 10 mV. (right) Corresponding QV relations for holding at 60, 0, and −90 mV are shown. Only a very modest mode shift is observed (P > 0.2). (B) Ionic currents of ShakerIR-A359C-I384A in response to a series of depolarizing pulses (50 ms) from a holding potential of −90 mV to voltages between −120 and 60 mV in steps of 10 mV. (right) Corresponding QV relations for holding at 0 and −140 mV and the GV (Vhold = −140 mV) are shown. The QVs are significantly shifted (P < 0.0001). (C) Gating currents of ShakerIR-W434F-F484A in response to a series of depolarizing pulses (50 ms) from a holding potential of −90 mV to voltages between −120 and 60 mV in steps of 10 mV. (right) Corresponding QV relations for holding at 0 and −90 mV are shown. They are not significantly shifted to one another (P > 0.7). (A–C) Data are shown as mean ± SD. (D) Gating and ionic currents of ShakerIR-F484A in response to depolarizing pulses from −90 mV to voltages between −120 and 60 mV in steps of 10 mV. (E) Effect of 100 mM TEA (top) and change in reversal potential to −30 mV in the presence of TEA (bottom; V = −70 mV). Only ionic current is affected, whereas gating currents remain the same.
	Figure 4. Mode shift in Shaker K+ channels. (A) FV curves for the three mutants ShakerIR-A359C, ShakerIR-A359C-I384N, and ShakerIR-A359C-F484G for holding potential (HP) at −90 and 0 mV in response to a series of depolarizing pulses to a potential of −120 to 60 mV and 40 to −160 mV, respectively. Reversal potential was adjusted to 0 mV. The FVs of A359C were significantly shifted (P < 0.0001), and the uncoupled mutants were not (P ≥ 0.3). (B) Comparison of FV and QV curves of the three mutants ShakerIR-A359C-W434F, ShakerIR-A359C-W434F-I384N, and ShakerIR-A359C-W434F-F484G for holding potentials at −90 and 0 mV in response to voltage pulses of 50-ms duration to voltages ranging between −160 and 50 mV in steps of 10 mV. (C) FV of ShakerIR-A359C from a holding potential of −90 mV to a prepulse of 0 mV for variable duration (0–1,200 ms) followed by a series of pulses of 50 to −160 mV in steps of 10 mV. The FV curves were fitted to a sum of two Boltzmann curves. (right) The fraction of the FV0 Boltzmann as a function of the duration of the depolarizing prepulse is shown. (A–C) Data shown are mean ± SD.
	Figure 5. Fluorescence signal indicating conformational changes of S4. (A) Fluorescence response to depolarizing pulses from −90 mV to voltages between−120 and 100 mV and 180 mV for ShakerIR-A359C and ShakerIR-A359C-I384N/F484G, respectively, for a duration of 100 ms. Channels are fluorescently labeled with tetramethyl-rhodamine maleimide at position A359C on top of the S4. (B) Time constants of exponential fits of the onset of the fluorescence signal obtained in A. (C) Comparison of fluorescence signals for saturating pulses of ShakerIR-A359C (V1/2 = −37.5 mV), ShakerIR-A359C-I384N (V1/2 = −55.4 mV), and ShakerIR-A359C-F484G (V1/2 = −56.8 mV). (D) QV of ShakerIR-W434F from a holding potential (HP) of −90 mV to a prepulse of 0 mV for variable duration (0–50 ms) followed by a series of pulses of 30 to −160 mV in steps of 10 mV. The QV curves were fitted to a sum of two Boltzmann curves. (middle) The shift of the V1/2 of the two Boltzmann distributions as a function of the duration of the depolarizing prepulse is shown. The shifts were fitted to single exponentials with time constants of τ = 4.0 and 8.8 ms for more positive and more negative ones, respectively. (right) The fraction of the QV0 Boltzmann as a function of the duration of the depolarizing prepulse is shown. (B and D) Data shown are mean ± SD.
	Figure 6. Alignment of Kv channels and energetics of mode shift. (A) Mode shift of the charge movement in ShakerIR and ShakerIR-W434F and relationship with the energies of pore opening and closing. (B) Alignment of the S4-S5 linker and C-terminal S6 region of human Kv channels. I384, F481, and F484 are highlighted.

References [+] :

Bao, Voltage-insensitive gating after charge-neutralizing mutations in the S4 segment of Shaker channels. 1999, Pubmed, Xenbase