Revealing a hidden conducting state by manipulating the intracellular domains in KV10.1 exposes the coupling between two gating mechanisms
- Oncophysiology Group. Max Planck Institute for Multidisciplinary Sciences, City Campus, Germany
- Neurophysics Laboratory, Göttingen Campus Institute for Dynamics of Biological Networks, Germany
Figures
Figure 1 with 1 supplement
Characterization of ΔPASCap and E600R mutants.
(A) Raw current traces resulting from depolarizations between –100 and +120 mV in WT (black), ΔPASCap (orange), and E600R (blue). The dashed lines indicate zero current. The stimulus protocol is …
-
Figure 1—source data 1
Stimulus voltage and individual current amplitudes related to Figure 1B.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig1-data1-v1.xlsx
-
Figure 1—source data 2
Stimulus voltage and individual rising times related to Figure 1D.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig1-data2-v1.xlsx
Figure 1—figure supplement 1
Structure of the cytoplasmatic ring of Kv10.1.
(A) Space-filling model from the side and from the cytoplasmatic side (bottom). The PAS domains are represented in orange, and the CNBHD is colored in blue. The rest of the protein is drawn in …
Figure 2
The biphasic GV corresponds to two sequential events.
(A) The GV of all tested mutants show biphasic behavior. (N: ∆2–10=6, ∆PASCap = 7, ∆eag = 7, E600R=11; mean ± SEM). All are well described by Equation 3b in a global fit with fixed parameters for …
-
Figure 2—source data 1
Stimulus voltage and individual current amplitudes related to Figure 2A.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig2-data1-v1.xlsx
-
Figure 2—source data 2
Stimulus voltage and individual current amplitudes related to Figure 2D.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig2-data2-v1.xlsx
Figure 3
Mg2+ induces a shift of the first component in the depolarizing direction.
(A) Raw current traces from oocytes expressing ∆eag 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 …
-
Figure 3—source data 1
Stimulus voltage and individual current amplitudes related to Figure 3A.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig3-data1-v1.xlsx
-
Figure 3—source data 2
Stimulus potential and individual values of activation time constant related to Figure 3B.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig3-data2-v1.xlsx
Figure 4
Alternating stimuli reveal larger macroscopic 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 …
Figure 5 with 1 supplement
Single ∆PASCap channels reveal longer open times at moderate depolarizations.
(A) Representative current traces at the indicated voltages obtained from a holding potential of –100 mV. (B) Comparison between traces obtained at +10 mV (light blue) and +40 mV (dark blue). The …
Figure 5—figure supplement 1
Single channel activity in an outside-out patch from an oocyte expressing ΔPasCap.
Traces were obtained by depolarizing from –100 mV to –60 mV in 10 s intervals. After 30 pulses in the control solution (60 mM extracellular KCl, black traces), the patch was exposed to 100 µM …
Figure 6
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, …
-
Figure 6—source data 1
Conditioning potential and individual rise times related to Figure 6B.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig6-data1-v1.xlsx
-
Figure 6—source data 2
Prepulse potential and amplitudes normalized to the –20 mV condition related to Figure 6C.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig6-data2-v1.xlsx
Figure 7
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 ∆PASCap (A) and E600R (B). (N: ∆PASCap = 7, E600R=6; mean ± SEM) …
-
Figure 7—source data 1
Stimulus potential and individual normalized amplitudes in ∆PASCap after prepulse voltages of –100 and –160 mV.
Related to Figure 7A.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig7-data1-v1.xlsx
-
Figure 7—source data 2
Stimulus potential and individual normalized amplitudes in E600R after prepulse voltages of –100 and –160 mV.
Related to Figure 7B.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig7-data2-v1.xlsx
-
Figure 7—source data 3
Stimulus potential and individual normalized amplitudes in ∆PASCap and ∆PASCapL322H.
Related to Figure 7C.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig7-data3-v1.xlsx
-
Figure 7—source data 4
Stimulus potential and individual normalized amplitudes in E600R and E600R L322H.
Related to Figure 7D.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig7-data4-v1.xlsx
Figure 8 with 4 supplements
CaM stabilizes O1.
(A) A transient rise in intracellular Ca2+ using 5 µM ionomycin and thapsigargin increases ∆PASCap current amplitude (in the absence of external chloride) and the IV relationship becomes linear …
-
Figure 8—source data 1
Stimulus potential and individual normalized amplitudes in ∆PASCap, ∆PASCapBDN ∆PASCapBDC2.
Related to Figure 8C.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig8-data1-v1.xlsx
-
Figure 8—source data 2
Stimulus potential and individual normalized amplitudes in E600R, E600RBDN, and E600RBDC2.
Related to Figure 8D.
- https://cdn.elifesciences.org/articles/91420/elife-91420-fig8-data2-v1.xlsx
Figure 8—figure supplement 1
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 …
Figure 8—figure supplement 2
The upper traces show the average of normalized ramps at the indicated times for ∆PASCap (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: ∆PASCap = 8, E600R=10). The lower panels correspond to the first derivative of the …
Figure 8—figure supplement 3
Like in Figure 8—figure supplement 2, the upper traces show the average of normalized ramps for ∆PASCapL322H.
(A) and E600RL322H (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: ∆PASCapL322H …
Figure 8—figure supplement 4
Ca2+ is required for efficient interaction between KV10.1 and CaM.
(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) …
Figure 9 with 5 supplements
A single model can reproduce all experimental observations.
Figure 9—figure supplement 1
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) (Zhang et …
Figure 9—figure supplement 2
The triphasic tail currents result from transient re-population of O1 during channel deactivation.
(A) Simulated currents from Figure 9B display simple, monophasic tail currents after weak depolarizations but triphasic tail current after +80 mV depolarization, similar to experimental results (see …
Figure 9—figure supplement 3
Current simulations for ∆PASCap upon depolarization to +20 and+80 mV after a conditioning pulse to –160 or –100 mV.
The current at +20 mV is larger after –160 mV, while the current at +80 mV is unaffected by the conditioning pulse (see Figure 6A).
Figure 9—figure supplement 4
Side-by-side comparison of experimental data and model prediction during depolarizations between –100 and +100 mV in the presence of high (60 mM) (A) or low extracellular [K+] (2.5 mM) (B).
The model describes the features of the experimentally observed currents, except the sustained rising phase during strong depolarizations.
Figure 9—figure supplement 5
Model variants can account for features of related channels and mutated variants.
(A) E600R gating features are consistent with slower flickering of a less populated O2. After lowering the stability of O2 (larger rclose/ropen and larger ζ/ε) and an overall slower flickering …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | Kv10.1 | NA | NCBI NM_002238.4 | |
| Antibody | anti-Myc | SIGMA | M4439, RRID:AB_439694 | 3 µg IP, 1:1000 immunoblot |
| Antibody | anti-Myc | Abcam | ab206486; RRID:AB_2861226 | 3 µg IP |
| Antibody | anti-Kv10.1 | Chen et al., 2011 | polyclonal anti-Kv10.1 | 1:1500 |
| Recombinant DNA reagent | pSGEM Kv10.1 | Addgene #85704 | ||
| Chemical compound, drug | Thapsigargin | Abcam | ab120286 | 5 µM |
| Chemical compound, drug | Ionomycin | Abcam | ab120116 | 5 µM |
| Chemical compound, drug | Astemizole | Esteve Química | N/A | 100 µM |
| Software, algorithm | Patch Master | HEKA | ||
| Software, algorithm | FitMaster | HEKA | ||
| Software, algorithm | Igor Pro | WaveMetrics |
Additional files
-
Supplementary file 1
Parameters of a global fit that linked the first component of the biphasic response.
- https://cdn.elifesciences.org/articles/91420/elife-91420-supp1-v1.docx
-
Supplementary file 2
Model parameters.
- https://cdn.elifesciences.org/articles/91420/elife-91420-supp2-v1.docx
-
Supplementary file 3
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.
- https://cdn.elifesciences.org/articles/91420/elife-91420-supp3-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/91420/elife-91420-mdarchecklist1-v1.pdf
