Noncanonical mechanism of voltage sensor coupling to pore revealed by tandem dimers of Shaker

Abstract

In voltage-gated potassium channels (VGKC), voltage sensors (VSD) endow voltage-sensitivity to pore domains (PDs) through a not fully understood mechanism. Shaker-like VGKC show domain-swapped configuration: VSD of one subunit is covalently connected to its PD by the protein backbone (far connection) and non-covalently to the PD of the next subunit (near connection). VSD-to-PD coupling is not fully explained by far connection only, therefore an additional mechanistic component may be based on near connection. Using tandem dimers of Shaker channels we show functional data distinguishing VSD-to-PD far from near connections. Near connections influence both voltage-dependence of C-type inactivation at the selectivity filter and overall PD open probability. We speculate a conserved residue in S5 (S412 in Shaker), within van der Waals distance from next subunit S4 residues is key for the noncanonical VSD-to-PD coupling. Natural mutations of S412-homologous residues in brain and heart VGKC are related to neurological and cardiac diseases.

Fig. 1

Biophysical properties of Shaker dimers when mutated in PDs. a Top: representation of Shaker protein showing wild-type voltage sensor (VSD^wt) and pore domains (PD^wt); Middle: Top view of Shaker channel in the membrane, showing each subunit (protein) of the tetramer represented by a different color, with N and C representing amino-terminus and carboxy-terminus, respectively. The black circle in the center represents K⁺ ions within the conductive pathway. Bottom: Typical K⁺ currents from Shaker channels expressed in Xenopus oocytes with the indicated voltage protocol. Horizontal bar means 2 s. b Top and middle: schematics showing dimerized Shaker, with protomer 1 in blue and protomer 2 in red. These channels do not contain W434F mutations and are called wt-PD Shaker dimers. Bottom: K⁺ currents recorded from wt-PD Shaker dimers in similar condition as in a. c, d Top and middle: representation of Shaker dimers containing the mutation W434F (W434F-Shaker dimers) in protomer 1 in c and in protomer 2 in d. The mutant domains are marked by a diamond. Bottom: K⁺ currents recorded with the respective channels in similar conditions as in a. In e–h, open squares are for Shaker, gray circles for wt-PD Shaker dimers, black triangles, and black squares for W434F-Shaker dimers with W434F mutation in protomers 1 and 2, respectively. e Normalized conductance–voltage (G–V) curves for the channels represented in a–d. f Time constants of the K⁺ currents inactivation in the indicated channels during 10 s depolarizing voltages periods (same protocol shown in a, bottom, and same symbols convention as in e). Fast and slow time constants from two exponentials fit to the data (see “methods” section for details), are shown for all Shaker dimers as in b–d. g Fast component relative amplitude of the exponential components in the channels indicated. h K⁺ currents 10 s inactivation curves (Inact-V) in the channels indicated. All data points are the average of 4–6 independent experiments. The vertical bars in e–h are the standard error of the mean. VSD^wtPD^wt: Shaker; VSD^wtPD^wt-VSD^wtPD^wt: wt-PD Shaker dimer; VSD^wtPD^W434F-VSD^wtPD^wt and VSD^wtPD^wt-VSD^wtPD^W434F: W434F-Shaker dimers with W434F in protomer 1 and in protomer 2, respectively

Fig. 2

Schematic representation of near and far configuration Shaker dimers. a Near configuration dimers shown in two possible designs, both with VSD^ILT and PD^W434F in different protomers (VSD^wtPD^W434F-VSD^ILTPD^wt or VSD^ILTPD^wt-VSD^wtPD^W434F). Protomer 1 is shown in blue and protomer 2 is shown in red in the top left and right panels with the mutant VSD indicated by circles and mutant PD indicated by diamonds. Note that mutant domains are in different protomers in both cases. b Far configuration dimers shown in their two possible designs, where VSD^ILT and PD^W434F are always in the same protomer (VSD^ILTPD^W434F-VSD^wtPD^wt or VSD^wtPD^wt-VSD^ILTPD^W434F). Protomers are color coded as in a. Similarly, the mutant VSD are indicated by circles and mutant PD indicated by diamonds

Fig. 3

Functional characterization of all types of W434F-Shaker dimers bearing VSD^ILT. K⁺ currents from dimers with PD^W434F mutation in protomer 1 are shown in a and b, configured as near and far channels, respectively. Horizontal bar in a, top, means 2 s. In c and d, K⁺ currents are shown from dimers with PD^W434F mutation in protomer 2, configured as near and far, respectively. All currents in a–d were activated by the same voltage protocol as shown in a, top panel. G–V and Inact-V curves are shown in e for near (orange) and far (green) dimers with W434F in protomer 1 and in f with W434F in protomer 2. Curves in black shown in e and f are from the corresponding control, PD^W434F in protomer 1 and in protomer 2, respectively, and with no VSD mutated (see Fig. 1). Inact-V curves in W434F-Shaker dimers, near and far, with the PD^W434F in protomer 1 (g) and in protomer 2 (h) are also shown together with the Q–V curve for VSD^ILT. i The shift in G–V curves introduced by the presence of VSD^ILT in Shaker (black bar) and in the four types of W434F-Shaker dimers, as labeled in the graph. j and k show K⁺ currents inactivation fast and slow time constants taken from two exponential fits (see “methods” section) from near and far configuration dimers, respectively. In both graphs filled and empty circles (orange or green) are for dimers with PD^W434F in protomer 1 and in protomer 2, respectively. l Fraction of the fast inactivating component are shown for near and far dimers with the position of the PD^W434F indicated by the labels in the graph. All data points are the average of 4–6 independent experiments. The vertical bars in e–l are the standard error of the mean

Fig. 4

Study of the VSD^mut//PD^W434F interfaces in W434F-Shaker dimers. a Voltage dependences of Shaker containing different VSD^mut. Plots show three voltage dependences of Shaker channels with different VSDs: V₀ and V₁ from Q–V and V_1/2 from G–V curves. b, c Inact-V curves from W434F-Shaker dimers, near and far configuration, respectively and as indicated, for each VSD^mut case. Mutations in the VSD are color/shape coded and are shown in the insets. d, e G–V curves for the same channels, near and far, shown in b and c with the mutations indicated by same color/shape code as in b and c. All Inact-V curves, except from near dimers containing VSD^361A or VSD^ILT, were fitted by a two-state model: noninactivated and inactivated (see “methods” section for details), generating a voltage-dependent parameter V_inact (Supplementary Tables 1 and 2). In near dimers containing VSD^361A or VSD^ILT, the curves were fitted by two independent two-state models to generate two voltage-dependent parameters: V_1st and V_2nd. f, g Plots of V_inact, V_1st, and V_2nd from near and far different dimers as labeled, respectively. Squared residuals from the fittings that generated the voltage-dependent parameters V_inact, V_1st, and V_2nd are also plotted and connected to them by the vertical dotted lines. Shifts in V_inact (or V_1st and V_2nd) from near (orange circles for V_inact, squares for V_1st and triangles for V_2nd, h and j) and far dimers (green circles, i and k) relative to the values in the respective Shaker dimers with VSD^wt only plotted against the respective shifts in V₀ (h and i) and V₁ (h and i) from VSD^mut taken from Q–V curves in nonconductive Shaker. l The same shift values of V_inact, V_1st and V_2nd shown in h–k from near and far dimers were plotted against the shifts in V_1/2 in Shaker induced by the respective VSD^mut (also relative to channels with VSD^wt only). Labels at each data point were omitted for clarity. All data points are the average of 4–6 independent experiments. The vertical bars in b–g are the standard error of the mean. The error bars in plots h–l were omitted for clarity

Fig. 5

wt-PD Shaker dimers (no W434F mutation) and nonconductive Shaker dimers (all PD mutated with W434F) show independent VSD domains. a Q–V curves are shown for all nonconductive dimers studied including dimers with only PD^W434F and the ones possessing two VSD^mut, indicated in the inset in a color/shape code. The curves were fitted with two independent two-state models for the generation of two voltage dependences values, V₀^* and V₁*. These values are plotted in b and for comparison, V₀ and V₁ (from nonconductive Shaker) are plotted in c. d G–V curves from wt-PD Shaker dimers possessing only VSD^wt and the ones possessing two VSD^mut, similarly indicated in the inset in a color/shape code. Data were fitted with a two-state model and the values of the G–V midpoints (V_1/2*) were used to calculate the shifts produced by the mutations in the VSD as compared with V_1/2* from wt-PD Shaker dimers that only contains VSD^wt (e). A horizontal dotted line shows the overall average value of the % shift and its value is 49.8%. All data points are the average of 4–6 independent experiments. The vertical bars in a and d are the standard error of the mean. The plots in b and c were omitted of error bars for clarity. Continuous lines over the data points represent the best fit of two independent two-state models for Q–V curves in a and a two-state model for G–V curves in d

Fig. 6

Putative key residue S412 in the VSD//PD interface in Shaker. The graph shows Q–V curves recorded in nonconductive Shaker, as well as G–V curves from Shaker carrying (red circles) or not (black circles) the mutation S412V, supposedly disruptive of the VSD//PD interface. Q–V curves are plotted as filled circles and G–V curves with open circles. Q–V curves were fitted with a three-state model to obtain V₀ and V₁ voltage dependences. G–V curves were fitted with a two-state model to get a midpoint V_1/2. All data points are the average of 4–6 independent experiments. The vertical bars are the standard error of the mean. Continuous lines over the data points represent the best fit of a three-state model for Q–V curves and a two-state model for G–V curves. All V₀, V₁, and V_1/2 voltage-dependent parameters are shown in Supplementary Table 2

Fig. 7

Structural models of a Shaker-like channel (K_V1.2, PDB:3LUT), showing the suggested VSD//PD interface. a Top view of the tetrameric channel, with two subunits in blue and red colors and the other two, for clarity, in light gray. Notice that the black square region indicates the VSD//PD interface between the VSD from different blue and red neighboring subunits. b Black square region from a zoomed in and turned by 90°. The cartoon shows with ribbons the S4 segment from one subunit in blue and the S5 segment from the neighbor subunit in red. Colored spheres are the van der Waals volumes of the atoms from the residues indicated (V369 in blue, S412 in green, S411 in orange, F433 in yellow, and finally W434 in magenta (residues numbering as in Shaker)). These residues are proposed to be the physical connection of the noncanonical coupling pathway between VSD and PD. c Top view of the zoomed black square region shown in a. Color code and labels correspond to a. Note that in a and in b the van der Waals surfaces are in contact, giving support to the hypothesis of the VSD//PD interface