Figures and data
Characterization of ΔPASCap and E600R mutants.
A. Raw current traces resulting from depolarizations between -100 and +120mV in WT (black), ΔPASCap (orange) and E600R (blue). B. GV plots corresponding to the three channel variants (colors as in A). (N: WT = 6, ΔPASCap = 7, E600R = 11; mean±SEM) C. Normalized traces to the indicated voltages to reveal the acceleration of activation with depolarization in the mutants. WT does not activate at -60mV type. D. Rise time of ΔPASCap (up) and E600R (down) as a function of voltage. The activation is much slower than in WT up to +50mV but reaches the speed of WT with stronger stimuli. E. Tail currents at -100 mV after depolarizations to potentials in the weak, medium, or strong range (up to down, see text for details). The arrows indicate the direction of the change in tail peak amplitude with increasing voltage. F. Normalized tail currents at -100 mV after depolarizations to the indicated voltages.
The biphasic GV corresponds to two sequential events.
A. The GV of all tested mutants show biphasic behavior. (N: Δ2-10 = 6, ΔPASCap = 7, Δeag = 7, E600R = 11; mean±SEM). All are well described by Eq. 4 in a global fit with fixed parameters for the first component. B. Distribution of the three components used for the fits as a function of voltage. C.A discontinuous form of Δ2-10 shows attenuated biphasic behavior. D. GV plots of Δ2-10 and Δ2-10.L341Split (N: Δ2-10 = 6, Δ2-10.L341Split = 5; ±SEM ). Split fitted using Eq. 4.
Mg2+ induces a shift of the first component in the depolarizing direction.
A. Raw current traces from oocytes expressing Δeag channels in response to depolarizations from a holding potential of -100 mV to voltages between -100 and +120 mV in the presence of 0, 1 or 5 mM MgCl2 in the external solution (legend in B). Scale bars are 1μA and 200 ms. B. Average GV plots (with SEM) obtained under the same conditions from n=6, 6, and 5 recordings (0, 1, and 5 mM MgCl2). The Vhalf of the first component shifts from -35.8 mV in the absence of extracellular Mg2+ to -27.9 and -9.9 mV in 1 mM and 5 mM MgCl2. C. Time constants of the early activation show decelerating effect of Mg2+. Symbols depict averages, vertical lines the range and horizontal lines individual experiments. For voltages, where conductances or time constants could not be reasonably estimated no data are displayed.
Alternating stimuli reveal larger conductance for O1.
A. Alternating potential between -80 and 80 mV in the WT results in current amplitudes that are smaller than those during a sustained stimulus (Upper left traces). In contrast, E600R gave rise to larger currents when the stimulus was intermittent and too short to allow occupancy of O2 (upper right). B. The effect was qualitatively similar for ΔPASCap, which consistently gave rise to larger current upon oscillating stimuli between -20 and +50 mV than during a constant pulse to +50 mV.
Hyperpolarization promotes access to a large conductance, slowly activating open state.
A. Raw current traces in response to the stimuli depicted in the scheme. B. The rise time to 80% of the maximal current during the depolarizing stimulus is plotted vs. prepulse voltage (N: WT = 7, ΔPASCap = 9, E600R = 8; mean±SEM). Although the activation is much slower for both mutants (note the different y axis for the mutants), they retain a strong dependence on the prepulse potential. C. Normalized end-pulse current (I/I-20) is plotted vs. prepulse voltage (N: WT = 10, ΔPASCap = 8, E600R = 8; mean±SEM). The amplitude of the current at +40 mV increased markedly when the holding potential was below -100 mV in the mutants, while the amplitude in WT changed only marginally.
Deep closed states facilitate access to O1.
A, B. Conditioning pulses to -160 mV potentiated the first component and hence the biphasic behavior of the I/V relationships for ΔPASCap (A) and E600R (B). (N: ΔPASCap = 7, E600R = 6; mean±SEM ) C, D. A mutation known to impair access to deep closed states (L322H) largely removes the initial phase of the GV curves for ΔPASCap (C) and E600R (D). (N: ΔPASCap = 7, ΔPASCapL322H = 6, E600R = 11, E600RL322H= 7; mean±SEM).
CaM stabilizes O1.
A. A transient rise in intracellular Ca2+ increases ΔPASCap current amplitude (in the absence of external chloride) and the IV relationship becomes linear (upper left traces). The lower traces represent the average normalized response comparing 0 and 60s. (ΔPASCap, light trace, N = 8, ΔPASCapL322H, dark trace, N= 5; the shadowed area indicates SEM). Right side traces: The same treatment in a channel carrying a mutation (L322H) reducing access to deep closed states results in the appearance of a biphasic IV upon Ca2+ rise. B. E600R behavior is comparable to ΔPASCap. Average normalized traces (E600R, light trace, N = 10; E600RL322H , dark trace, N = 11; the shadowed area indicates SEM) are represented in the lower panel. C. Mutation of the C-terminal CaM binding domain (BDC2) in ΔPASCap reduces strongly the first component of the biphasic GV, while deletion of the N-terminal binding site (BDN) did not have any effect (N: ΔPASCap = 7, ΔPASCapBDN = 7, ΔPASCapBDC2 = 7; mean ± SEM). D. The reduction of the first component in E600R when the C-terminal CaM binding site is mutated is also present but less intense. (N: E600R = 11, E600RBDN = 9, E600RBDC2 = 11; mean±SEM)
A single model can reproduce all experimental observations.
A. In response to an I-V-protocol (Fig. 1A), the model displays biphasic activation, more clearly represented in the derived G-V curve (right, compare to Fig. 1B). The filled area indicates the contributions of the two open states. The tail-currents show a complex dependence on test-pulse voltage and time. B. Two traces from A, shown with longer repolarization. The first 100 ms of the tail currents are displayed on an extended timescale. The colored areas indicate the contributions of the two open states. After weak depolarization to -20mV, the tail-currents originate almost entirely from the mutant-specific open state O1 (blue area). After strong depolarization, O2 mediated currents initially dominate (light grey). The early, rapid decrease in current amplitude results from closure of O2. During a delay phase, increasing current through an increasingly populated O1 compensate for this closure, until eventually O1 (dark grey) also closes, but with a much slower kinetics. C. In response to 10 ms pulses alternating between -20 and +50 mV, the model shows currents that exceed the currents obtained with constant pulses to +50 mV (compare Fig. 4 B). Relating the currents during the positive and negative pulses to the concurrent currents elicited by the two constant pulses (brown and orange), the ratio lies by about 1.6 for the period starting 200 ms after the pulse onset. This excess current depends sensitively on the duration and voltage of the two pulse components. The right series of simulations displays the results for 15 ms pulses to -20 mV, alternating with 15 ms pulses to voltages from 15mV to 65 mV. The corresponding responses to the constant pulses are displayed with thin dotted lines. To facilitate perception of the excess current, the five groups of traces are scaled individually, so that the peak amplitude of the dotted response elicited by the stronger depolarizations is displayed at equal size throughout. The vertical scalebars correspond to the same absolute current. From top to bottom, the excess current ratios at 200 ms changes are 1.05, 1.38, 1.74, 1.46, and 1.16. D. The binding of Ca2+-CaM is implemented through change in the activation energy, corresponding to a shift in the equilibrium voltage of the gating transitions. The decomposition of the current into the individual open states’ contribution shows that for increasing voltage shifts – representing high [Ca2+]i – the mutant-specific O1 closes later into the ramp, until eventually all current is carried by O1.
Chloride currents obtained upon treatment with ionomycin plus thapsigargin. The increase in cytosolic Ca2+ translates in a Cl- current that can be used to estimate the amplitude and duration of the Ca2+ increase. Notice that Ca2+ returns to basal levels in approximately 150 s. The gray area indicates SEM. N=9
The upper traces show the average of normalized ramps at the indicated times for ΔPASCap (A) and E600R (B) after induction of Ca2+ rise. At 60, 150 and 300 s, the dashed line corresponds to the ramp at time 0. The shadowed area indicates SEM (N: ΔPASCap = 8, E600R = 10). The lower panels correspond to the first derivative of the current traces to illustrate the changes in slope.
Like in Supplement 2, the upper traces show the average of normalized ramps for ΔPASCapL322H (A) and E600L322H (B) at the indicated times after induction of Ca2+ rise. At 60, 150 and 300 s, the dashed line corresponds to the ramp at time 0. The shadowed area indicates SEM (N: ΔPASCapL322H = 5, E600RL322H = 11). The lower panels correspond to the first derivative of the current traces to illustrate the changes in slope.
A. Ca2+-dependent binding of CaM to KV10.1. CaM was precipitated with anti-Myc antibody and the resulting pulled down fraction was immunoblotted using polyclonal anti-KV10.1 antibody. B Quantification of all experiments reveals a strong dependence on Ca2+ concentration in WT and to a similar extent in mutant channels. The fraction of KV10.1 pulled down increases by two orders of magnitude in high Ca2+.
Cartoon depicting the state model proposed.
A two-layer Markov-Model depicting possible conformations for the sensor and the ring. The sensor in each subunit can independently adopt one of three conformations (up, middle, and down) (Han et al., 2023). The up conformation represents the active conformation. The conformation of the ring changes across the two layers. The second conformation in the upper layer allows the channel to access “O2” provided that all sensors are in the up conformation. Transitions within each layer is governed by (α, β, γ, δ), we proposed that the transitions are identical in the two layers. Transitions between the two levels are governed by the rate constants κ and λ
Current simulations for ΔPASCap upon depolarization to -20 or +80 mV in the presence of high extracellular K+. A. The triphasic current kinetics (see Fig. 1E) observed experimentally at +80 mV (insert) is predicted by the model. B. Occupancy of State_4 (state closest to O1), O1 and O2 as a function of time during a depolarization to +80 mV.
Current simulations for ΔPASCap upon depolarization to +20 and +80 mV after a conditioning pulse to -160 or -120 mV. The current at +20 mV is larger after -160 mV, while the current at +80 mV is unaffected by the conditioning pulse (see Fig. 6A)
Current simulations for ΔPASCap upon alternating stimuli between -20 and +50 mV, as compared to sustained depolarization to each of the potentials (see Fig. 4B)
Current simulations for ΔPASCap under ramp depolarization using shifted rate constants (γ, δ, κ) to mimic the effect of CaM binding (see Fig. 7A)
Side-by-side comparison of experimental data and model prediction during depolarizations between -100 and +100 mV in the presence of high (A) or low extracellular K+ (B). the model describes the features of the current except the sustained rising phase during strong depolarizations.
Parameters of a global fit that linked the first component of the biphasic response
parameters describing the voltage dependence of the transitions between states in the Markov model of KV10.1 PASCap, in conjunction with Eq. 1.
Primers used for infusion cloning or site-directed mutagenesis.
The sequences are listed 5’-3’. For mutagenesis primers, only the sense sequences are given. The reverse primers corresponded to the reverse-complement sequence.
