Molecular basis of the PIP2-dependent regulation of CaV2.2 channel and its modulation by CaV β subunits

Figures

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  Figure 1.. 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 while CaV α2δ subunit is mostly localized at the extracellular surface of the channel protein (Gao et al., 2021). Schematic model of CaV2.2 channels with plasma membrane (PM)-anchored β2a, cytosolic β2a(C3,4S) and β2c, or N-terminus engineered PM-recruited β2c (right). (B) Representative confocal images of tsA-201 cells expressing the PM marker Lyn-mCh and β2 isoforms or its derivatives fused to GFP without the α1 and α2δ1 subunits. Scale bar, 5 μm. The scatter plot shows a 2D intensity histogram of the red (Lyn-mCh) and green (β2-GFP) pixels in the confocal image. The value indicates the Pearson’s correlation coefficient (R) that is obtained by the Colocalization Threshold plugin of Fiji software (Image J). (C) Summary of Pearson’s coefficient between Lyn-mCh and the β2 construct (n = 10–11). (D) Current inactivation of CaV2.2 channels with β2 isoforms or its derivatives was…

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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 replaced with serine (gray). Lyn anchored to the plasma membrane via the myristoylation and palmitoylation modification sequence Lyn11 (blue). Orange indicates flexible linker between Lyn and β2c subunit. (B) Representative confocal images of tsA-201 cells expressing plasma membrane marker Lyn-mCh and β2 engineered derivatives fused to GFP in the presence of α1 and α2δ1 subunits. Scale bar, 5 μm. The scatter plot shows a 2D intensity histogram of the red (Lyn-mCh) and green (β2-GFP) pixels in the confocal image. The text indicates the Pearson’s correlation coefficient (R) values. (C) Summary of Pearson’s coefficient between the Lyn-mCh and each β2 engineered derivatives with α1 and α2δ1 subunits. Dots indicate the individual data points for each cell (n = 8–11). Data are mean ± standard error of the mean (SEM). No significant difference among the cells. Figure 1—figure…

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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 perfused with 0.1 mM GTP without Dr-VSP expression (bottom). Peak tail current is indicated by arrowheads (trace a, black head; trace b, red head). (B) Summary of the CaV2.2 current inhibition (%) by a depolarizing pulse to +120 mV in cells without Dr-VSP (n = 6). (C) Current inhibition by a 120-mV-depolarizing pulse was recorded in cells intracellularly perfused with 1 mM GDP-β-S without (top) or with (bottom) Dr-VSP expression. (D) Summary of the CaV2.2 current inhibition (%) by depolarizing pulse without (top) and with (bottom) Dr-VSP expression (n = 8–11). Dots indicate the individual data points for each cell. Data are mean ± standard error of the mean (SEM). ***p < 0.001, using one-way analysis of variance (ANOVA) followed by Tukey post hoc test. Figure 1—figure supplement 2—source data 1.Current inhibition (%) by a depolarizing pulse in cells in the absence of Dr-VSP. Figure…

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  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 pulses to 10 mV in cells with chimeric Lyn-linker-β2c derivatives. Data of Figure 1C from Lyn-β2c (gray trace) are reproduced to facilitate visual comparison. (C) Summary of CaV2.2 current inactivation with Lyn-linker-β2c derivatives (n = 9–12). Summary data of Figure 1E from Lyn-β2c (white bar) are reproduced to facilitate visual comparison. The fraction of current remaining after 100-ms depolarization (r100) to +10 mV. (D) Current inhibition of CaV2.2 channels with chimeric Lyn-linker-β2c derivatives by Dr-VSP-mediated PIP2 depletion. The traces before (a) and after (b) the strong depolarizing pulse to 120 mV were superimposed. Peak tail current is indicated by arrowheads (trace a, black head; trace b, red head). (E) Summary of CaV2.2 current inhibition (%) by PIP2 depletion in Dr-VSP-expressing cells (n = 9–12). Summary data of Figure 1E from Lyn-β2c…

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  Figure 2.. 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 tyrosine 406, lysine 408, and threonine 410 residues in the GK domain are replaced with alanine. Schematic model of CaV2.2 channels with engineered β2 constructs in which the SH3–GK intramolecular interaction is disrupted. Lyn-(∆N)β2: Lyn-labeled N-terminus-deleted β2 construct. Lyn-(∆N)β2 Mut: Lyn-(∆N)β2 construct with a disrupted SH3–GK intramolecular interaction. Inset: confocal images of tsA-201 cells expressing engineered β2 constructs labeled with mCherry without α1B and α2δ1 subunits. Scale bar, 5 μm. (B) Representative currents of CaV2.2 channels with engineered β2 constructs. The currents were measured during 500-ms test pulses to +10 mV (top). Current traces before (a) and after (b) a + 120-mV depolarizing pulse in cells expressing CaV2.2 channels with engineered β2 constructs and Dr-VSP (bottom).…

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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 trace) are reproduced for visual comparison. Data are mean ± standard error of the mean (SEM) (n = 8–11). Figure 2—figure supplement 1—source data 1.Current–voltage (I–V) curve of CaV2.2 current.

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  Figure 3.. 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 the GK domain, respectively. The new constructs were tagged with Lyn11 (RF or Lyn-FRB-Hook-GK-FKBP) to be tethered to the PM. Rapamycin (Rapa) addition triggers the formation of a tripartite FRB–rapamycin–FKBP complex, resulting in the movement of the FKBP domain to the PM (right). For Förster resonance energy transfer (FRET) imaging, chimeric β constructs labeled with YFP in the C-terminus and PM-targeting Lyn-CFP were coexpressed. Right, schematic model of CaV2.2 channels with RF before and after rapamycin application. Rapamycin induces the formation of the tripartite complex, resulting in a shift of the FKBP domain to the PM and an enhanced FRET signal. (B) Time courses of CaV2.2 currents (blue traces) and FRET ratio (green traces) were measured simultaneously in single cells expressing CaV2.2 channels with Cont (left), RF (middle), or RCF (right) and the membrane marker…

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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 obtained before and 30 s after rapamycin addition. (B) Summary of the rapamycin effects on the peak current amplitude of CaV2.2 with Cont, RF, or RCF. Rapamycin application increased the current amplitude only in channels with RF construct. Dots indicate the individual data points for each cell (n = 6–7). ***p < 0.001, using one-way analysis of variance (ANOVA) followed by Tukey post hoc test. Data are mean ± standard error of the mean (SEM). 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.. 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 sequences of Lyn (Lyn11 plus 12 aa linker) and the additional linkers are listed. (B) Current inactivation of CaV2.2 channels with diverse CaV β-GK derivatives was measured during 500 ms test pulses to +10 mV. (C) Effects of Dr-VSP-mediated PIP2 depletion on CaV2.2 channels with GK domain derivatives. Peak tail current is indicated by arrowheads (trace a, black head; trace b, red head). (D) Summary of current inactivation (blue bars; n = 9–12) and inhibition (%) by PIP2 depletion (red bars; n = 8–10) in CaV2.2 channels with CaV β GK derivatives. Data are mean ± standard error of the mean (SEM). Dots indicate the individual data points for each cell. (E) Normalized mean current inactivation and mean current inhibition by PIP2 depletion versus additional linker length (aa) of CaV β GK derivatives measured in…

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  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 full amino acid sequence of the linker is indicated in Figure 4A.

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  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 indicate the individual data points for each cell (n = 9–10). The online version of this article includes the source data, Figure 4—figure supplement 2—source data 1. 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 (blue trace, right). Holding potential is −80 mV and dashed line is zero current. (B) Population current density versus voltage relations for CaV2.2 channels with β2a (green trace) and β2c (blue trace). (C) Summary of current density (pA/pF) of CaV2.2 channels with β2a and β2c (n = 8–9). Cells were transfected with the same amount of cDNA. (D) Representative Ba2+ current traces elicited between −50 and +40 mV in 10 mV steps (see pulse protocol) in cells expressing CaV2.2 channels with Lyn-β2c (orange trace, left) and Lyn-48aa-β2c (purple trace, right). Holding potential is −80 mV and dashed line is zero current. (E) Population current density versus voltage relations for CaV2.2 channels with Lyn-β2c (orange trace) and Lyn-48aa-β2c (purple trace). (F) Summary of current density (pA/pF) of CaV2.2 channels with Lyn-β2c and Lyn-48aa-β2c (n = 7). Cells were transfected with the same amount of cDNA. Dots indicate the individual data…