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Figure 1. Schematic for Kv motions and kinetics. (A) Section through a bilayer-embedded Shaker in conformations C, CA, C4AP, and O (closed, closed-but-activated, closed-4subunits-activated-plus, open). First, consider the bilayer and the bilayer-protein interface; details of the bilayer's laterally acting forces on Kv channels are unknown, but as Fig 4 of Morris and Laitko (2005) and associated references point out, bilayer forces contribute to conformational equilibria. Insofar as the bilayer thins with stretch (dashed lines in each leaflet) the channel–lipid interface will change (two possibilities are depicted: accommodation on the left, mismatch on the right). Next, the protein conformations themselves. The vertical dotted lines are for positional reference. A closed-resting state (C) is depicted with the voltage sensors in a position stabilized by hyperpolarization. In the closed-activated (CA) states favored by depolarization, the sensors and neighboring domains repack (the motions that yield C1A–C4A are largely independent), but the gates remain shut. Each C→CA subsumes greater complexity (e.g., to describe Shaker WTIR and ILT gating current, two independent steps in series are needed; Ledwell and Aldrich, 1999), which we ignore here. Next are two concerted motions. First four voltage sensors do a final motion together (yielding “activated-plus”). The next concerted step is opening of the tetrameric gate, C4AP→O. B summarizes this as a kinetic scheme showing the voltage-dependent steps. In ILT, the concerted voltage-dependent forward rate C4A→C4AP is rate limiting (Del Camino et al., 2005), whereas in Shaker 5aa, we found (Laitko and Morris, 2004) that independent voltage-dependent steps that we take to be C→CA are rate limiting. In WT channels, the two rates are similar, making it harder to distinguish activation from pore opening.
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Figure 2. ILT kinetics. (A) Averaged (n = 15 runs) currents from one patch. The membrane was stepped from −90 mV to the first and then to the second voltage indicated. Current activation and decline were fitted with single exponentials. (B) τ(V) from exponential fits and g(V) from tail current amplitudes, averaged results from 16 and 11 patches, respectively, with SEM, and fitted with Eqs. 1 and 3. Both τ(V) and g(V) can be fitted with these expressions over their entire respective voltage ranges, but with slightly different z and K0 that cannot be brought into agreement for the two datasets. Dashed lines: best τ(V) fit (zα = 1.0, z = 2.0, α0 = 0.58 s−1, K0 = 740) and Eq. 3 for g(V) with these parameters. However in the voltage range of ILT activation (60–170 mV, solid lines), z and K0 from the g(V) fit (z = 1.9, K0 = 810) describe τ(V) very well (zα = 1.2, α0 = 0.25 s−1).
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Figure 3. Stretch and ILT currents. Here and for other figures, black (lines or filled circles) = before stretch, red (lines or squares) = during stretch, gray (lines or diamonds) = after stretch. Data from a sample patch. (A) Averaged sample currents (n = 5 runs; repeats were done in succession before moving to another voltage), for steps from −90 mV to the indicated voltage and back to 20 mV, with axis breaks to better visualize the tail. For the leftmost set of currents, an arrow marks the start of the depolarizing step. In all sets, a time scale is provided by labels at t = 0 and at the first major tick (“0” marks the start of recording, not the beginning of a step; the initial flat section is the holding current at −90 mV). Below, at right, for three voltages, tail currents are shown on expanded time scales, as indicated. (B) g(V) relation from tail current amplitudes after stepping back to 20 mV. The fit with Eq. 3 shows that gating charge is unaffected by stretch, whereas K0 increased (z = 1.9, K0 = 700/1170/720 for before/during/after). (C) Averaged τ(V) (seven patches) and normalized g(V) (four patches) with and without stretch, fits with Eqs. 1 and 3 in the range of activation voltages. z and K0 from the g(V) fit (z = 2.0, K0 = 780 without and 1260 with stretch) describe τ(V) very well (zα = 1.3, α0 = 0.18 s−1 without and 0.11 s−1 with stretch).
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Figure 4. Stretch and other Shaker mutants. Sample currents taken before, during, and after stretch at a range of voltages for (A) Shaker WT (fast N-type inactivation present), (B) Shaker WTIR, and (C) Shaker 5aa. (D) Average normalized g(V) (n = 4) from peak tail current amplitudes, with and without stretch, for WTIR. Fits with a fourth order Boltzmann (Eq. 3 to the fourth power). z = 3.3 for both curves, K0 = 0.00093 and 0.00062 for without stretch and with stretch, respectively. The resulting total gating charge of ∼13 (i.e., 4 × 3.3) is in agreement with the literature. Determining the g(V) with the tail current method is problematic for WT and 5aa, as they possess similar time scales of activation and inactivation, however it is clear from the sample currents in A and C that their g(V) relations would be left shifted, too. (Colors and symbols as in Fig. 3).
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Figure 5. ILT tail currents. (A) Averaged sample currents (n = 10) with exponential fits (fits and data overlap). (B) Tail current time constant vs. voltage on a logarithmic scale. Exponential fit: τ0 = 2.7 ms, z = 1. The data would be situated on the left flank of the τ(V) relation in Fig. 2 B, z here is equivalent to zβ. (C) Averaged samples (n = 3) from a tail current family recorded from a patch with stretch-accelerated tails. (D) τ(V) for that patch, exponential fits: τ0 = 3.3/2.3/ 3.3 ms for before/during/after stretch, z = 0.95 for all.
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Figure 6. Resolvable tail currents at various voltages. WTIR tail current τ(V) in normK (n = 8) (gray squares) and hiK (n = 6) (black inverted triangles) compared with the ILT tail τ(V) (n = 9) in normK (gray open circles) from Fig. 1 B. Double exponential fit for WTIR hiK: τ0,1 = 170 ms, z1 = 1.3, τ0,2 = 4.2 ms, z2 = 0.46. Due to a discontinuity near EK, WTIR normK cannot be fitted in its entirety; the shallow and steep branches were fitted with single exponentials (shallow branch: z = 0.67, τ0 = 25 ms, steep branch: z = 1.1, τ0 = 300 ms). ILT tail τ(V) is from Fig. 1 B (single exponential z = 0.92, τ0 = 3.6 ms). For net K+ movement being inward, WTIR closing was faster; the lack of a shallow branch or a τ(V) jump in ILT makes sense if the discontinuity (in WTIR normK) depended on a change in current direction.
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Figure 7. WTIR and stretch. (A) Stretch-slowed tail currents (recorded in hiK) samples and τ(V) with double exponential fit: τ0,1 = 34 ms, z1 = 0.78, τ0,2 = 3.6 s, z2 = 2.8 without stretch, τ0,1 = 62 ms, z1 = 0.88, τ0,2 = 3.4 s, z2 = 2.8 with stretch. (B) Stretch-accelerated WTIR tail currents recorded in normK; τ(V) cannot be fitted with double exponentials. Fits with two separate lines: left branch (before and during [after cannot be fitted]) τ0 = 5.9/4.0 ms, z = 0.44/0.44; right branch (before, during, after): τ0 = 52/25/38 s, z = 0.93/0.85/0.85.
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Figure 8. Effects of stretch on Shaw2 F335A. (A i) Shaw2 F335A macroscopic current (single traces) during a multistep protocol (voltages indicated in mV; protocol rationale is given in the text), before, during, and after stretch (using −25 mm Hg). An expanded inset reveals that current onset was sigmoid shaped. (A ii) From the same patch, stretch difference currents at the indicated levels of applied suction (difference currents = [(before + after)/2 – during]). (B) From an oocyte expressing Shaw2 F335A at a low level, (B i) current at 0 mV (10 traces averaged; membrane continuously at 0 mV, using −30 mmHg) before, during, and after stretch. From the same patch (but using −35 mm Hg), (B ii) excerpts from single traces, and (B iii) all-points amplitude histograms from eight runs per condition (i.e., before/during/after stretch) during steps from −100 to 0 mV show that the outward (=upward) unitary current jump amplitude (6.5 pA) was stretch independent. Since for this patch, linear subtraction was not done, interference from the capacitative current was avoided by omitting the first 200 ms of each run for ii and iii.
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Figure 9. Kv1 structure cartoons. (A i) A cartoon modified from Pathak et al. (2005) depicts the last concerted motion of the voltage sensor exerting a laterally acting (expansion) force within the protein. Subsequently, a concerted relaxation pulls open the channel gate (presumably this expands the hinge region). A ii suggests that the last charge concerted movements could, alternatively, couple to forces that constrict or compact the closed channel (“spring extension”) before a concerted gate region expansion (“spring relaxation”) that opens the pore. To provide context with the kinetic schemes, A iii shows the last two steps from Fig. 1 A. Note that if C4AP is the most expanded state, this is not the factor dominating the stretch responses of ILT (this would not predict the observed stretch deceleration of ILT activation, and it would predict decreased ILT gmax with stretch, which was not observed). As explained in the text, B (modified from Webster et al., 2004) depicts the shutter-like action of the S6 pore hinge as seen from the intracellular space (rings signify the Cd bridges used to locate gating-related helix–helix interactions). The finding of Pathak et al. (2005) that a small concerted S4 movement (<15% of total sensor motion) impels an opening expansion is indicated. The cartoon in C centers on a schematic of an open-like Kv1.2 channel as seen from the extracellular side, adapted from Long et al. (2005a). A curved arrow points to the junction of sensor and pore modules in the primary structure. A perimeter line highlights the fact that the interface (i.e., amino acid residues interacting with bilayer lipid molecules on the z axis) is more extensive than for, say, a cylindrical tetramer. Bilayer stretch would alter the z axis LPP along this entire x,y perimeter. Surface active molecules (white and gray pentamers) whose mobility dropped at loci on the channel perimeter (gray molecules) could alter LPPs and hence conformational equilibria in excess of what would be predicted solely from the line tension due to bulk (white) molecules (Ly and Longo, 2004), making some distinctions between low-affinity binding effects and bilayer mechanical effects largely semantic.
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