Molecular basis of the PIP2-dependent regulation of CaV2.2 channel and its modulation by CaV β subunits
- Department of Brain Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Republic of Korea
Figures
Figure 1 with 3 supplements
Current inactivation and PIP2 sensitivity in N-type CaV2.2 channels with different subtypes of the β2 subunit.
(A) Schematic diagram of high-voltage-activated (HVA) calcium channel complex viewed from the intracellular side (left). CaV β subunit is located beside the domain II of α1B in the cytosolic side …
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Figure 1—source data 1
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in N-type CaV2.2 channels with different subtypes of the β2 subunit.
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Figure 1—figure supplement 1
Subcellular localization of N-terminus engineered constructs of β2 subunit in the presence of α1 and α2δ1 subunits.
(A) Comparison of the N-terminal sequences of N-terminus engineered β2 construct. In the palmitoylation-resistant mutant β2a(C3,4S), the palmitoylation residues cysteine 3 and 4 (purple) are …
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Figure 1—figure supplement 1—source data 1
Pearson’s coefficient between Lyn-mCh and the β2 construct in the presence of α1 and α2δ1 subunit.
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Figure 1—figure supplement 2
Summary of the CaV2.2 current inhibition by a 120-mV-depolarizing pulse in cells without or with Dr-VSP.
(A) Schematic diagram showing the Dr-VSP activation by depolarizing pulse protocol (top). CaV2.2 currents before (a) and after (b) the depolarizing pulse are superimposed in cells intracellularly …
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Figure 1—figure supplement 2—source data 1
Current inhibition (%) by a depolarizing pulse in cells in the absence of Dr-VSP.
- https://cdn.elifesciences.org/articles/69500/elife-69500-fig1-figsupp2-data1-v1.xlsx
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Figure 1—figure supplement 2—source data 2
Summary of the CaV2.2 current inhibition by Dr-VSP-mediated PIP2 depletion in cells were recorded with pipette solution containing GDP-β-S.
- https://cdn.elifesciences.org/articles/69500/elife-69500-fig1-figsupp2-data2-v1.xlsx
Figure 1—figure supplement 3
Effects of inserting a flexible linker between Lyn and β2c subunit on current inactivation and PIP2 sensitivity of CaV2.2 channels.
(A) Amino acid sequences of inserting a linker (orange) between Lyn11 (blue) and N-terminus of β2c subunit (black). (B) Representative currents of CaV2.2 channels were measured during 500-ms test …
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Figure 1—figure supplement 3—source data 1
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in CaV2.2 channels with chimeric Lyn-linker-β2c derivatives.
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Figure 2 with 1 supplement
Disruption of SH3–GK interaction in the plasma membrane (PM)-recruited CaV β2 subunit leads to an increase in both current inactivation and PIP2 sensitivity of CaV2.2 channels.
(A) Left, a diagram showing how the SH3–GK intramolecular interaction is disrupted in β2 constructs (top). Phenylalanine 92, histidine 94, arginine 107, and valine 109 residues in the SH3 domain and …
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Figure 2—source data 1
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in N-type CaV2.2 channels with the engineered β2 construct.
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Figure 2—figure supplement 1
Disruption of the SH3–GK intramolecular interaction of β2 subunit does not shift current–voltage (I–V) curve of CaV2.2 current.
Normalized peak current–voltage (I–V) relations of CaV2.2 currents evoked by voltage steps to the indicated potential (mV) in cells with β2 engineered derivatives. Data from Lyn-(∆N)β2 WT (gray …
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Figure 2—figure supplement 1—source data 1
Current–voltage (I–V) curve of CaV2.2 current.
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Figure 3 with 1 supplement
Effects of the real-time translocation of the GK domain to the plasma membrane (PM) on CaV2.2 channel gating.
(A) Left, a schematic diagram showing rapamycin-induced translocatable β2 chimeric constructs. Translocatable β2 chimeric constructs were invented by fusing FRB or FKBP to the N- and C-termini of …
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Figure 3—source data 1
Time courses of CaV2.2 currents and Förster resonance energy transfer (FRET) ratio.
- https://cdn.elifesciences.org/articles/69500/elife-69500-fig3-data1-v1.xlsx
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Figure 3—source data 2
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in CaV2.2 channels with rapamycin-induced translocatable β2 chimeric constructs before and after rapamycin.
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Figure 3—figure supplement 1
The real-time translocation of the GK domain to the plasma membrane increased the current amplitude of CaV2.2 channels.
(A) CaV2.2 currents before (black trace) and during (red trace) the rapamycin application in cells expressing CaV2.2 channels with Cont (left), RF (middle), or RCF (right). The currents were …
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Figure 3—figure supplement 1—source data 1
Relative peak current amplitudes of CaV2.2 channels with chimeric Lyn-linker-β2c derivatives.
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Figure 4 with 4 supplements
Flexible linker length between Lyn and the GK domain of the β subunit performs a key role in determining both the current inactivation and the PIP2 sensitivity of CaV2.2 channels.
(A) Schematic diagram of diverse flexible linkers (∆N) inserted between Lyn and GK (G) domain. The length of each linker is calculated by the worm-like chain (WLC) model (see Methods). Amino acid …
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Figure 4—source data 1
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in CaV2.2 channels with the engineered β2 GK derivatives.
- https://cdn.elifesciences.org/articles/69500/elife-69500-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
IUPRED web-server result of inserted linker.
Red line is disorder tendency of the linker and blue dotted line is 0.5. Higher disorder tendency (>0.5) suggests that the linker is intrinsically disordered protein and random coil structure. The …
Figure 4—figure supplement 2
Summary of time constants for CaV2.2 current activation.
Time constants of current activation in CaV2.2 channels with diverse CaV β-GK derivatives were measured from the data shown in Figure 4B. Data are mean ± standard error of the mean (SEM). Dots …
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Figure 4—figure supplement 2—source data 1
Time constants of current activation in CaV2.2 channels with diverse CaV β-GK derivatives.
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Figure 4—figure supplement 3
Current density in N-type CaV2.2 channels with β2 variants.
(A) Representative Ba2+ current traces elicited by voltage steps from −50 and +40 mV in 10 mV steps (see pulse protocol) in cells expressing CaV2.2 channels with β2a (green trace, left) and β2c …
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Figure 4—figure supplement 3—source data 1
Population current density versus voltage relations for CaV2.2 channels with β2 variants.
Peak current density at 10 mV (pA/pF) of CaV2.2 channels with β2 variants.
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Figure 4—figure supplement 4
Flexible linker length between Lyn and GK domain of β subunit is important in determining the current density and the voltage-dependent gating of CaV2.2 channels.
(A) Population current density versus voltage relations for CaV2.2 channels with diverse flexible linker inserted between Lyn and GK constructs. Data from Lyn-GK (gray trace) are reproduced to …
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Figure 4—figure supplement 4—source data 1
Population current density versus voltage relations and the voltage dependence of normalized steady-state activation for CaV2.2 channels with the engineered β2 GK derivatives.
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Figure 5 with 3 supplements
Polybasic motif at the C-terminal end of the I–II loop influences determination of steady-state activation, current inactivation, and PIP2 sensitivity of CaV2.2 channels.
(A) Schematic diagram of phospholipid-binding residue-neutralizing mutations within the C-terminal end of the I–II loop in the α1B subunit. The phospholipid-binding residues (R465, R466, K469, and …
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Figure 5—source data 1
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in cells expressing α1B WT and 4A mutants with β2a and β2c.
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Figure 5—figure supplement 1
Sequence alignment of the C-terminal end of the I–II loop in CaV α1 subunits.
Putative PIP2-binding residues within the C-terminal end of I–II loop were highlighted in red. The calcium channel subunits α1S of rat CaV1.1, α1C of rat CaV1.2, α1D of human CaV1.3, α1F of human CaV…
Figure 5—figure supplement 2
Current inactivation and PIP2 sensitivity of mutant CaV2.2 channels with Lyn-β2c and Lyn-48aa-β2c.
(A) Current inactivation was measured during 500-ms test pulses to +10 mV in cells expressing α1B WT (black traces) and 4A (red traces) with Lyn-β2c (left) or Lyn-48aa-β2c subunit (right). (B) …
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Figure 5—figure supplement 2—source data 1
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in CaV2.2 channels with Lyn-β2c and Lyn-48aa-β2c.
- https://cdn.elifesciences.org/articles/69500/elife-69500-fig5-figsupp2-data1-v1.xlsx
Figure 5—figure supplement 3
Neutralization of polybasic residues in the distal end of the I–II loop domain plays a crucial role in determining current inactivation and PIP2 sensitivity of CaV2.2 channels with β2c.
(A) Schematic diagram of the C-terminal end of the I–II loop in CaV α1B subunits. Two positive amino acids (R465 and R466) were neutralized to alanine residues (α1B R465,466A) and two positive amino …
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Figure 5—figure supplement 3—source data 1
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in cells expressing WT α1B, α1B R465,466A, and α1B R476,477A with β2a and β2c.
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Figure 6 with 1 supplement
Modulation by M1 muscarinic stimulation and Dr-VSP activation in Gβγ-insensitive chimeric α1C-1B CaV2.2 channel.
(A) Schematic diagram showing the inhibitory signaling from M1 muscarinic acetylcholine receptor (M1R) and Dr-VSP to Gβγ-insensitive chimeric α1C-1B channel. VI, voltage-independent inhibition; VD, …
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Figure 6—source data 1
Current inhibition (%) of α1C-1B WT and 4A mutants by M1R or Dr-VSP activation in cells expressing with β2a and β2c.
Summary of the prepulse experiments in before and Oxo-M perfused cells with α1C-1B WT and 4A mutants with β2a or β2c subunits.
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Figure 6—figure supplement 1
Polybasic motif at the C-terminal end of the I–II loop affects in determining the M2 muscarinic modulation of CaV2.2 channels.
(A) Schematic diagram showing the M2 muscarinic acetylcholine receptor (M2R)-mediated inhibitory signaling to CaV2.2 channels. VD, voltage-dependent inhibition. (B) Current inhibition by M2R …
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Figure 6—figure supplement 1—source data 1
Current inhibition (%) of α1B WT and 4A mutants by M2R activation in cells expressing with β2a and β2c.
Summary of the prepulse experiments in before and Oxo-M perfused cells with α1B WT and 4A mutants with β2a or β2c subunits.
- https://cdn.elifesciences.org/articles/69500/elife-69500-fig6-figsupp1-data1-v1.xlsx
Figure 7 with 1 supplement
PIP2-binding residues within the S4II domain plays an important role in determining steady-state activation and PIP2 sensitivity of CaV2.2 channels.
(A) Distance analysis of PIP2-binding site in the S4II domain of α1B subunit. Two amino acids (R584 and K587) interacting with the 5-phosphate of PIP2 were neutralized to alanine residues (RA/KA). (B…
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Figure 7—source data 1
Current inactivation (r100), current inhibition (%) by PIP2 depletion and the V1/2 of normalized steady-state activation in cells expressing WT α1B, WT α1B RA/KA, 4A α1B, and 4A α1B RA/KA with β2a or β2c.
- https://cdn.elifesciences.org/articles/69500/elife-69500-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
PIP2 sensitivity of α1B R578,581A with β2a or β2c subunits.
(A) Schematic diagram of two positive amino residues (R578 and R581) replaced with alanine residues (α1B R578,581A) within S4II domain of α1B subunit. (B) Current inhibition by Dr-VSP-mediated PIP2 …
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Figure 7—figure supplement 1—source data 1
Current inhibition (%) by PIP2 depletion in cells expressing WT α1B and α1B R578,581A with β2a or β2c.
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Figure 8
Schematic model showing the differential regulation of CaV2.2 channels with membrane-anchored and cytosolic β subunits by PIP2.
The channel possesses two distinct PIP2-interacting sites: the PIP2-binding pocket in the S4II domain and the nonspecific phospholipid-biding site in the I–II loop C-terminus. When the CaV2.2 …
Author response image 1
Summary of the Cav2.
2 current inhibition by Dr-VSP-mediated PIP2 depletion in cells were recorder with pipette solution containing GDP-β-S. (A) Cells received a test pulse (a), then a depolarisation of 120mV, a …
Additional files
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Supplementary file 1
Primers for β2 chimera constructs.
- https://cdn.elifesciences.org/articles/69500/elife-69500-supp1-v1.xlsx
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Supplementary file 2
Primers for deletion or mutagenesis of CaV α1B and β2 constructs.
- https://cdn.elifesciences.org/articles/69500/elife-69500-supp2-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69500/elife-69500-transrepform1-v1.pdf
