(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 …
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in N-type CaV2.2 channels with different subtypes of the β2 subunit.
(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 …
Pearson’s coefficient between Lyn-mCh and the β2 construct in the presence of α1 and α2δ1 subunit.
(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 …
Current inhibition (%) by a depolarizing pulse in cells in the absence of Dr-VSP.
Summary of the CaV2.2 current inhibition by Dr-VSP-mediated PIP2 depletion in cells were recorded with pipette solution containing GDP-β-S.
(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 …
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in CaV2.2 channels with chimeric Lyn-linker-β2c derivatives.
(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 …
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in N-type CaV2.2 channels with the engineered β2 construct.
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 …
Current–voltage (I–V) curve of CaV2.2 current.
(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 …
Time courses of CaV2.2 currents and Förster resonance energy transfer (FRET) ratio.
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in CaV2.2 channels with rapamycin-induced translocatable β2 chimeric constructs before and after rapamycin.
(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 …
Relative peak current amplitudes of CaV2.2 channels with chimeric Lyn-linker-β2c derivatives.
(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 …
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in CaV2.2 channels with the engineered β2 GK derivatives.
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 …
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 …
Time constants of current activation in CaV2.2 channels with diverse CaV β-GK derivatives.
(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 …
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.
(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 …
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.
(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 …
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in cells expressing α1B WT and 4A mutants with β2a and β2c.
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…
(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) …
Current inactivation (r100) and current inhibition (%) by PIP2 depletion in CaV2.2 channels with Lyn-β2c and Lyn-48aa-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 …
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.
(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, …
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.
(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 …
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.
(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…
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.
(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 …
Current inhibition (%) by PIP2 depletion in cells expressing WT α1B and α1B R578,581A with β2a or β2c.
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 …
Primers for β2 chimera constructs.
Primers for deletion or mutagenesis of CaV α1B and β2 constructs.