Figure 1. Thin filament C- and B-state structures drawn from PDB 7UTI and 7UTL coordinates. (A) The C-state, high-Ca²⁺ filament. (B) The B-state, low-Ca²⁺ filament. (C) The high and low-Ca²⁺ filaments superposed. All panels display the solvent-excluded surface of actin subunits rendered to only show the front-facing strand of actin for clarity (actin subunits colored light blue, light grey). The pointed ends of actin are shown facing up. Troponin and tropomyosin are presented in ribbon format in A–C. Color scheme: TnI, navy blue; TnC, orange; TnT, yellow; tropomyosin in its high-Ca²⁺ position, gold; and at low-Ca²⁺, magenta. The troponin core domain, where TnI, TnC, and TnT converge, points toward the barbed end of the filament. Note: Left-facing arrows point to the navy blue TnI C-terminal domain extension on the outer actin domains in B; also note the apparent movement of the magenta low-Ca²⁺ tropomyosin strand toward TnI. (A–C) Salt bridges between Lys 328 on actin (blue spheres) and acidic residues on tropomyosin (red spheres) are highlighted in A and B by right-handed arrows; again note that a residue on inner tropomyosin α-helices contact K328 of each actin subunit shown (canonical salt-bridge contacts listed in Table 1 ; other actin–tropomyosin contacts are provided in Fig. S2 ). The TnI C-terminal domain, which is released from TnC at low-Ca²⁺, is seen to bind to tropomyosin by means of salt bridges (indicated by left-handed arrows in B as well as by red and navy blue spheres in B and C). Non-polar interactions of TnI with tropomyosin and actin are not highlighted here but are discussed in Lehman et al. (2021) . (D) The two-component α-helical tracks of coiled-coil tropomyosin that are defined by the high- and low-Ca²⁺ tropomyosin trajectories in C are rendered as gold and magenta threads and superposed on actin. In C and D, sites marked by arrowed brackets show regions where one set of α-helical chains of the superposed high- and low-Ca²⁺ tropomyosin coiled coils coincide within a 2-Å cutoff, while the rest diverge. This is most easily seen in D by noting the overlaying threads. This positional commonality extends over contiguous tropomyosin residues 65–74, 103–109, 148–151, 188–192, 233–244, and 262–269, located in pseudorepeats 2–7; the tropomyosin head-to-tail junction is not depicted here, but is detailed in Pavadai et al. (2020a) ; Pavadai et al. (2020b) ; and Risi et al. (2022) . (E) The superhelical tracts defined by high- and low-Ca²⁺ tropomyosin are rendered here as gold and magenta threads, respectively, and represent the average center of mass of low- and high-Ca²⁺ tropomyosin on actin (magenta and gold, respectively); note the distance between the magenta and gold threads, which are furthest apart over middle actin subunits shown, i.e., between tropomyosin pseudorepeats 3–5, and they are closest together between C-terminal tropomyosin residues 248 and 265 (indicated by broad arrowed brackets at the bottom of the panel). Actin residues 328 are highlighted as blue spheres in D and E for reference. (F–H) Magnified versions of B–D focused on regions in which the C-terminal domain of TnI interacts with actin and tropomyosin. More about this image found in Thin filament C- and B-state structures drawn from PDB 7UTI and 7UTL co...

Figure 2. TnI-induced pivoting of tropomyosin. Transverse sections of C- and B-state thin filament structures shown in Fig. 1 viewed here over C-terminal TnI residues R192 (A–C) and R170 (E–G); these TnI residues localize in close proximity to tropomyosin E114 and E139. (A and E) C-state sections (B and F), B-state sections, (C and G), B- and C-state sections superposed (same color representation as in Fig. 1 ). (D and H) Corresponding dynamics of tropomyosin and TnI shown in superposed sections. (A–C) Sections highlight tropomyosin pivoting, and in B and C salt-bridge formation between tropomyosin E114 (red spheres) and TnI residue R192 (navy blue spheres). Note that in C, superposition of B- and C-state sections shows inner tropomyosin helices overlapping (short arrow), while TnI appears to have drawn the outer tropomyosin helix toward the B-state position (bold arrow). (E–G) Sections highlight tropomyosin pivoting and salt-bridge formation of tropomyosin E138 with actin residue K328 (blue on actin surface), and in F and G, a salt bridge between tropomyosin E139 with TnI residue R170 (navy blue spheres). Note that in G, the superposition of B- and C-state sections shows inner tropomyosin helices are closely apposed (short arrow), while TnI appears to attract tropomyosin E139 of the inner tropomyosin helix (oblique bold arrow) here, causing the outer helix to pivot toward the B-state position (horizontal bold arrow). (D and H) Representation of tropomyosin and TnI dynamics on actin observed during MD simulations viewed in sections in D corresponding to ones in A and B and in H to E and F. The data are from the last 12 ns of simulations and represent 109 frames of B-state and 77 frames of C-state structures superposed. (D) Representative positions of the charged side-chain atoms of tropomyosin residue E114 (magenta and gold points for low- and high-Ca²⁺ data, respectively) and TnI residue R192 (navy blue points) taken during MD simulations of B- and C-state thin filament models (magenta and gold points show the positions of negatively charged Glu OE1 and OE2 atoms on E114, respectively, and navy blue points for positively charged Arg atoms NH1, NH2, and NE on R192). (H) Analogously rendered positions of negatively charged side-chain atoms on tropomyosin residue E139 and positively charged atoms on TnI residue R170 during MD simulations again performed on B- and C-state filaments (same color scheme as above); points assumed by positively charged NZ atoms actin residue K328 in H also shown for reference (light blue). The red and cyan spheres in D and H show the respective COO⁻ and NH₃⁺ side-chain positions of tropomyosin and TnI at the beginning of the MD simulations; the blue sphere in Fig. 2 H shows the initial location of the terminal K328 NH₃⁺ at the onset of MD. The curved arrows indicate the direction of tropomyosin movement toward the actin outer domain (myosin steric-blocking position) associated with tropomyosin–TnI complex formation as in C and G. More about this image found in TnI-induced pivoting of tropomyosin. Transverse sections of C- and B-state...

Figure S2. Measurement of electrostatic interaction between actin and tropomyosin along thin filaments. (A and B) Electrostatic interaction energy between individual tropomyosin residues and actin residues 326 and 328 were plotted for atomic models of C- and B-state filaments (A; PDB IDs 7UTI ... More about this image found in Measurement of electrostatic interaction between actin and tropomyosin alon...

Figure 3. C-state to B-state pivoting of tropomyosin. Transverse sections through superposed 7UTI C-state and 7UTL B-state thin filament models but only with the tropomyosin positions displayed for comparison (gold, C-state tropomyosin; magenta, B-state tropomyosin). Selected slab sections s... More about this image found in C-state to B-state pivoting of tropomyosin. Transverse sections through su...

Figure 1. Single muslce fiber fibertyping. (a) A representative image of a single muscle fiber, post–Mant-ATP chase experiment, which has undergone fibertyping with MyHC β-slow/type I antibody and has stained positively, depicting that is a type I muscle fiber. (b) A representative image of a ... More about this image found in Single muslce fiber fibertyping. (a) A representative image of a single mu...

Figure 2. Myosin head conformation in type II myofibers is shifted in PA individuals compared to sedentary individuals. (a) A representative Mant-ATP chase experiment decay graph showing exponential decay of type I and type II single muscle fibers from sedentary individuals and PA individuals. (b and c) The percentage of myosin heads in skeletal myofibers in the DRX (b) and SRX (c). This was estimated from the equation shown in the Materials and methods section. (d) T1 value in seconds denoting the ATP turnover lifetime of the DRX. (e) T2 value in seconds denoting the ATP turnover lifetime in the SRX. Each colored triangle data point represents the mean value of all fibers from each subject. Statistical significance was calculated using Student’s t test, P < 0.05 was taken to be significant. n = 5–6 individuals per subject group. More about this image found in Myosin head conformation in type II myofibers is shifted in PA individu...

Figure 3. DRX myosin ATP turnover time is changed in the myofibers of elite-endurance athletes but not SA s. (a) A representative Mant-ATP chase experiment decay graph showing exponential decay of type I single muscle fibers from endurance athletes and type I and type II single muscle fibers from strength athletes. (b and c) The percentage of myosin heads in skeletal myofibers in the DRX (b) and SRX (c). This was estimated from the equation shown in the Materials and methods section. (d) T1 value in seconds denoting the ATP turnover lifetime of the DRX. (e) T2 value in seconds denoting the ATP turnover lifetime in the SRX. Each colored triangle data point represents the mean value of all fibers from each subject. Statistical significance was calculated using Student’s t test, P < 0.05 was taken to be significant. n = 5–6 individuals per subject group. More about this image found in DRX myosin ATP turnover time is changed in the myofibers of elite-endurance...

Figure 1. Structure of the pore region and the four-state gating model of Shaker-IR. (A) The structure of the pore domain of Shaker-IR channel (amino acid residues 390-489) in open conformation (PDB accession no. 7SIP ) visualized by PyMol. The S5, S6, pore helix, and pore loop segments of two diagonally opposed subunits are shown as ribbon representations with front and rear subunits removed for clarity. Colored amino acids are T449 (red), A471 (purple), L472 (cyan), and 473 (blue). Potassium ions in the selectivity filter are illustrated by black dots. (B) Helical wheel projection of the pore domain (residues 469–479). Amino acid residues indicated by * at the right side of the helical wheel are facing the water-filled cavity in the open state. Color-coding indicates amino acid characteristics, yellow: hydrophobic; gray: polar. (C) Composite gating states and a simplified four-state gating model in which four major gating states can be distinguished depending on the status of the activation (lower) and inactivation (upper) gates. Among the closed (C), open (O), open-inactivated (OI), and closed-inactivated (CI) states, only the open state is conducting. Arrows indicate the theoretically existing transitions between these gating states. RFI represents recovery from slow inactivation. More about this image found in Structure of the pore region and the four-state gating model of Sha...

Figure 2. Characterization of K ⁺ currents of the T449A/A471C Shaker-IR mutant. (A) Ionic currents of T449A/A471C channel expressed in tsA201 cells. The inside-out patch was held at −100 mV and depolarized to test potentials ranging from −80 to +70 mV in steps of 10 mV every 60 s. The duration of the depolarizing pulses was 1 s. Inset shows K⁺ currents evoked by the depolarizing pulses to the indicated membrane potentials. (B) Normalized G–V data (see Materials and methods) were obtained from the peak currents (I_peak) at the test potential of E_m and the K⁺ reversal potential (E_K) using G(V) = I_peak/(E_m – E_K). The G(V) values were normalized for the maximum conductance (G/G₀) for six independent measurements, averaged, and then plotted as a function of test potential. The superimposed solid line is the best-fit Boltzmann function to the averaged data points. The mean values ± SEM of the midpoint (V_1/2) and slope factor (s) are shown. (C) Activation kinetics were studied using 5-ms-long depolarizing pulse from a holding potential of −120 to +50 mV. Current traces were fitted using the Hodgkin-Huxley n⁴-model, and the activation time constant (τ_act) was used to describe the activation kinetics. The inactivation time constant (τ_i) of the current at +50 mV was determined by fitting a single exponential function to the decaying part of the currents shown in Fig. 2 A . Violin plots show Q1, median, and Q3 for n = 23 independent experiments, and symbols indicate individual data points. (D) Voltage dependence of steady-state inactivation was measured in inside-out patches. The voltage protocol (top) consisted of a brief (5 ms) step from a holding potential of −120 to +50 mV to record I_−120. The patch was then held between −110 and −20 mV in steps of 10 mV for 3 s, and then a short (5-ms) test pulse to +50 mV was applied to obtain I. After each pulse pair, the patches were held at −120 mV holding potential for 45 s. The fraction of noninactivated channels (I/I₋₁₂₀) at each prepulse potential was averaged for n = 5 cells (biological replicates) and plotted as a function of prepulse potential. The superimposed solid line shows the best-fit Boltzmann function to the averaged data points. The mean values ± SEM of the midpoint (V_1/2) and slope factor (s) are shown. (E) Kinetics of recovery from inactivation were determined using pairs of depolarizing pulses from the holding potential of −120 to +50 mV for 400 ms. The ipi at −120 mV varied between 1.8 and 60 s. Current traces highlighted in red, green, and blue were recorded during the second pulse with ipi of 1.8 and 60 s and control, prior to the start of the pulse pairs, respectively. (F) FR was calculated as I₂ − I_ss1/I₁ − I_ss1, where I₂ and I₁ are the peak currents during the second and first pulse, respectively, and I_ss1 is the current at the end of the first depolarization. The FR averaged for n = 5 cells versus ipi plot was fit with an exponential rise to maximum function to give the time constant of recovery from inactivation (τ_rec). Data are given as mean ± SEM (n = 5), n values are biological replicates in all panels. More about this image found in Characterization of K ⁺ currents of the T449A/A471C Sh...