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Inhibition <t>of</t> <t>Cav2.1</t> Ca2+ currents by the R1279X EA2-truncated form of Cav2.1 protein. A, Schematic cartoon representing the main Cav2.1 constructs used in this study. B, The Ca2+ current density in HEK293 cells expressing the Cav2.1 channel protein. The cells were cotransfected with plasmids encoding the human Cav2.1 subunit, the β1b subunit, and the α2/δ1 subunit. Mean ± SEM values of the current density for cells expressing the Cav2.1 subunit alone (white bar; n = 18) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22) are presented (***p < 0.001; Student's t test). The inset shows representative current traces at TP −40 and 0 mV [holding potential (HP), −80 mV]. C, Same experiments as in B performed in the neuroblastoma cell line NG108-15. The HP was −50 mV (***p < 0.001; Student's t test). Cav2.1 subunit alone (white bar; n = 15) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22). D, Measurements of native T-type and HVA Ca2+ current densities in R1279X-transfected NG108-15 cells. The T-current density (left) was measured in proliferative NG108-15 cells (Prolif.; 2–3 d after transfection; n = 9) and in differentiated NG108-15 cells (Diff.; 6 d after transfection; 3–4 d after switching to differentiation medium; n = 11) using HP −100 mV and TP −30 mV. The HVA current density, mainly corresponding to L- and N-type channel activities, was measured using HP −50 mV and TP 0 mV (right).

Gfp Ca V 2.1, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels"

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2844-07.2008

Inhibition of Cav2.1 Ca2+ currents by the R1279X EA2-truncated form of Cav2.1 protein. A, Schematic cartoon representing the main Cav2.1 constructs used in this study. B, The Ca2+ current density in HEK293 cells expressing the Cav2.1 channel protein. The cells were cotransfected with plasmids encoding the human Cav2.1 subunit, the β1b subunit, and the α2/δ1 subunit. Mean ± SEM values of the current density for cells expressing the Cav2.1 subunit alone (white bar; n = 18) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22) are presented (***p < 0.001; Student's t test). The inset shows representative current traces at TP −40 and 0 mV [holding potential (HP), −80 mV]. C, Same experiments as in B performed in the neuroblastoma cell line NG108-15. The HP was −50 mV (***p < 0.001; Student's t test). Cav2.1 subunit alone (white bar; n = 15) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22). D, Measurements of native T-type and HVA Ca2+ current densities in R1279X-transfected NG108-15 cells. The T-current density (left) was measured in proliferative NG108-15 cells (Prolif.; 2–3 d after transfection; n = 9) and in differentiated NG108-15 cells (Diff.; 6 d after transfection; 3–4 d after switching to differentiation medium; n = 11) using HP −100 mV and TP −30 mV. The HVA current density, mainly corresponding to L- and N-type channel activities, was measured using HP −50 mV and TP 0 mV (right).

Figure Legend Snippet: Inhibition of Cav2.1 Ca2+ currents by the R1279X EA2-truncated form of Cav2.1 protein. A, Schematic cartoon representing the main Cav2.1 constructs used in this study. B, The Ca2+ current density in HEK293 cells expressing the Cav2.1 channel protein. The cells were cotransfected with plasmids encoding the human Cav2.1 subunit, the β1b subunit, and the α2/δ1 subunit. Mean ± SEM values of the current density for cells expressing the Cav2.1 subunit alone (white bar; n = 18) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22) are presented (***p < 0.001; Student's t test). The inset shows representative current traces at TP −40 and 0 mV [holding potential (HP), −80 mV]. C, Same experiments as in B performed in the neuroblastoma cell line NG108-15. The HP was −50 mV (***p < 0.001; Student's t test). Cav2.1 subunit alone (white bar; n = 15) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22). D, Measurements of native T-type and HVA Ca2+ current densities in R1279X-transfected NG108-15 cells. The T-current density (left) was measured in proliferative NG108-15 cells (Prolif.; 2–3 d after transfection; n = 9) and in differentiated NG108-15 cells (Diff.; 6 d after transfection; 3–4 d after switching to differentiation medium; n = 11) using HP −100 mV and TP −30 mV. The HVA current density, mainly corresponding to L- and N-type channel activities, was measured using HP −50 mV and TP 0 mV (right).

Techniques Used: Inhibition, Construct, Expressing, Mutagenesis, Transfection

Effect of R1279X mutant on surface expression of Cav2.1 channel. A, HEK293 cells were cotransfected with the HA-tagged wild-type Cav2.1 subunit alone (with free GFP) or together with the R1279X mutant (fused to GFP). Left column, GFP fluorescence. Middle column, Staining of cells with monoclonal rat anti-HA antibody (primary antibody) and Alexa594 (secondary antibody). NP, Nonpermeabilized; P, permeabilized. Images were acquired using a Leica SP2 confocal microscope, with a 63× oil immersion objective. B, Luminometric ELISA assays to quantify surface expression of the HA-tagged Cav2.1 in the presence of the R1279X mutant and auxiliary subunits (**p < 0.01, ***p < 0.001; Student's t test). C, Western blots performed on HEK293 cells expressing Cav2.1-HA alone (−) or in the presence of R1279X (+) without (left) or with (right) auxiliary subunits. β1b-HA was used in these experiments (right).

Figure Legend Snippet: Effect of R1279X mutant on surface expression of Cav2.1 channel. A, HEK293 cells were cotransfected with the HA-tagged wild-type Cav2.1 subunit alone (with free GFP) or together with the R1279X mutant (fused to GFP). Left column, GFP fluorescence. Middle column, Staining of cells with monoclonal rat anti-HA antibody (primary antibody) and Alexa594 (secondary antibody). NP, Nonpermeabilized; P, permeabilized. Images were acquired using a Leica SP2 confocal microscope, with a 63× oil immersion objective. B, Luminometric ELISA assays to quantify surface expression of the HA-tagged Cav2.1 in the presence of the R1279X mutant and auxiliary subunits (**p < 0.01, ***p < 0.001; Student's t test). C, Western blots performed on HEK293 cells expressing Cav2.1-HA alone (−) or in the presence of R1279X (+) without (left) or with (right) auxiliary subunits. β1b-HA was used in these experiments (right).

Techniques Used: Mutagenesis, Expressing, Fluorescence, Staining, Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot

Channel misfolding and instability induced by the R1279X mutant. A, B, Pulse-chase experiments were performed on HEK293 cells 48 h after transfection (see Materials and Methods). Cells were labeled with 35S-methionine-cysteine for 20 min. The chase was performed for 0, 1, 2, or 4 h as indicated. After lysis, the Cav2.1 subunit was immunoprecipitated with an anti-HA antibody. The arrow indicates the band corresponding to the EA2 mutant that coimmunoprecipitates with the wild-type Cav2.1 subunit. In B (left), the β1b is detected (arrow). C, Quantification of three independent experiments using GE Healthcare software. D, Pulse-chase analyses performed on HEK293 cells transfected with CD4-AAXX alone or in the presence of R1279X. Quantification was performed as described above. E, Immunoprecipitations performed on cells coexpressing various subunit arrangements (as indicated) in the absence or presence of an untagged R1279X mutant (right) using standard SDS-PAGE (6%). Western blots (WBs) were performed with the indicated antibodies. F, Immunoprecipitations were performed between β1b-HA and GFP-R1279X as described in E. MW, Molecular weight.

Figure Legend Snippet: Channel misfolding and instability induced by the R1279X mutant. A, B, Pulse-chase experiments were performed on HEK293 cells 48 h after transfection (see Materials and Methods). Cells were labeled with 35S-methionine-cysteine for 20 min. The chase was performed for 0, 1, 2, or 4 h as indicated. After lysis, the Cav2.1 subunit was immunoprecipitated with an anti-HA antibody. The arrow indicates the band corresponding to the EA2 mutant that coimmunoprecipitates with the wild-type Cav2.1 subunit. In B (left), the β1b is detected (arrow). C, Quantification of three independent experiments using GE Healthcare software. D, Pulse-chase analyses performed on HEK293 cells transfected with CD4-AAXX alone or in the presence of R1279X. Quantification was performed as described above. E, Immunoprecipitations performed on cells coexpressing various subunit arrangements (as indicated) in the absence or presence of an untagged R1279X mutant (right) using standard SDS-PAGE (6%). Western blots (WBs) were performed with the indicated antibodies. F, Immunoprecipitations were performed between β1b-HA and GFP-R1279X as described in E. MW, Molecular weight.

Techniques Used: Mutagenesis, Pulse Chase, Transfection, Labeling, Lysis, Immunoprecipitation, Software, SDS Page, Western Blot, Molecular Weight

EA2 missense mutants act in a dominant-negative manner by misfolding and instability induction. A, Histograms of the mean Ca2+ current density (± SEM) obtained in a representative batch of NG108-15 cells expressing wild-type subunit alone (Cav2.1-HA, n = 18); the EA2 mutants alone (Cav2.1-HA-G293R, n = 33, 5 with detectable current; Cav2.1-HA-AY1593/94D, n = 6); and the wild-type and EA2 mutants together (Cav2.1-HA+Cav2.1-HA-G293R, n = 23, 3 with detectable current; Cav2.1-HA+Cav2.1-HA-AY1593/94D, n = 10). These experiments were conducted in the presence of the auxiliary β1b and α2/δ1 subunits. B, Normalized native T-type Ca2+ current densities (mean ± SEM) in the NG108-15 cells analyzed in A. The differences are not statistically significant between the various conditions tested (Student's t test). C, Western blots with the indicated antibodies (WB) were performed on HEK293 cells coexpressing Cav2.1-HA alone or in the presence of G293R and AY1593/94D mutants together with auxiliary subunits. The bottom panel shows the Western blot from cells expressing missense mutants alone. D, Pulse-chase analyses were performed on HEK293 cells transfected with wild-type Cav2.1 alone or in the presence of G293R. Quantification of three independents experiment was performed as described above. MW, Molecular weight.

Figure Legend Snippet: EA2 missense mutants act in a dominant-negative manner by misfolding and instability induction. A, Histograms of the mean Ca2+ current density (± SEM) obtained in a representative batch of NG108-15 cells expressing wild-type subunit alone (Cav2.1-HA, n = 18); the EA2 mutants alone (Cav2.1-HA-G293R, n = 33, 5 with detectable current; Cav2.1-HA-AY1593/94D, n = 6); and the wild-type and EA2 mutants together (Cav2.1-HA+Cav2.1-HA-G293R, n = 23, 3 with detectable current; Cav2.1-HA+Cav2.1-HA-AY1593/94D, n = 10). These experiments were conducted in the presence of the auxiliary β1b and α2/δ1 subunits. B, Normalized native T-type Ca2+ current densities (mean ± SEM) in the NG108-15 cells analyzed in A. The differences are not statistically significant between the various conditions tested (Student's t test). C, Western blots with the indicated antibodies (WB) were performed on HEK293 cells coexpressing Cav2.1-HA alone or in the presence of G293R and AY1593/94D mutants together with auxiliary subunits. The bottom panel shows the Western blot from cells expressing missense mutants alone. D, Pulse-chase analyses were performed on HEK293 cells transfected with wild-type Cav2.1 alone or in the presence of G293R. Quantification of three independents experiment was performed as described above. MW, Molecular weight.

Techniques Used: Dominant Negative Mutation, Expressing, Western Blot, Pulse Chase, Transfection, Molecular Weight

Endoplasmic reticulum retention and proteasomal degradation of the dominant-negative Cav mutants. A, Confocal images of nonpermeabilized NG108-15 cells expressing EA2 mutants and truncated Cav3.2 subunits. Alexa 594-coupled CT was used as plasma membrane marker (0.5 μg/ml). B, Confocal images of immunofluorescence staining performed on permeabilized NG108-15 cells expressing indicated EA2 mutants and truncated Cav3.2. Polyclonal anti-protein disulfide isomerase (Assay Designs, Ann Arbor, MI) was used as ER marker. C, Pulse-chase experiments were performed as in Figure 3 on cells transfected with EA2 mutants (R1279X and G293R) and Cav3.2 truncated forms. Chase was done for the indicated time (hours) with or without MG-132 proteasome inhibitor (50 μm). After lysis, the truncated Cav channels were immunoprecipitated with anti-GFP antibody. D, Representative pulse chase performed on HEK293 cells transfected with Cav2.1 and the R1279X mutant and the corresponding quantification (n = 3). During the chase, cells were treated with MG-132 (50 μm), leupeptin (20 μm), and NH4Cl (10 mm).

Figure Legend Snippet: Endoplasmic reticulum retention and proteasomal degradation of the dominant-negative Cav mutants. A, Confocal images of nonpermeabilized NG108-15 cells expressing EA2 mutants and truncated Cav3.2 subunits. Alexa 594-coupled CT was used as plasma membrane marker (0.5 μg/ml). B, Confocal images of immunofluorescence staining performed on permeabilized NG108-15 cells expressing indicated EA2 mutants and truncated Cav3.2. Polyclonal anti-protein disulfide isomerase (Assay Designs, Ann Arbor, MI) was used as ER marker. C, Pulse-chase experiments were performed as in Figure 3 on cells transfected with EA2 mutants (R1279X and G293R) and Cav3.2 truncated forms. Chase was done for the indicated time (hours) with or without MG-132 proteasome inhibitor (50 μm). After lysis, the truncated Cav channels were immunoprecipitated with anti-GFP antibody. D, Representative pulse chase performed on HEK293 cells transfected with Cav2.1 and the R1279X mutant and the corresponding quantification (n = 3). During the chase, cells were treated with MG-132 (50 μm), leupeptin (20 μm), and NH4Cl (10 mm).

Techniques Used: Dominant Negative Mutation, Expressing, Marker, Immunofluorescence, Staining, Pulse Chase, Transfection, Lysis, Immunoprecipitation, Mutagenesis

Image Search Results

The R1667P mutation occurs at the R4 position of the Repeat IV S4 voltage-sensing α-helix. ( a ) Schematic representation of Ca V 2.1 with Green Fluorescent Protein (GFP) fused to the amino-terminus (GFP-Ca V 2.1). The R to P substitution at residue 1667 is indicated by the red star. ( b ) Sequence comparison of the Repeat IV S4 helices of all known human Ca V 2.1 variants ( cf . accession no. NP_001120693.1), human Ca V 2.1 with the R to P substitution at the R4 position and human Ca V 2.2 (accession no. NM_001243812). Basic residues in positions R2-R6, as defined by Ca V 2.2 Cryo-EM structure are shown in green and the R4 R to P substitution is shown in red. ( c , d ) AlphaFold2 modeling of the voltage-sensing module of Repeat IV of human Ca V 2.1 . The S1–S4 helices are viewed from the lateral aspect ( c ) and from an extracellular ( d ) vantage points. Potential hydrogen bonds between R1667 and N1579 in S1, T1606 in S2, and S1641 in S3 are indicated by the blue dashed lines. F1609 (i.e., the gating charge transfer center) is also labelled. Panels ( c ) and ( d ) were published with permission https://creativecommons.org/licenses/by/4.0/ ( e ) Missense 3D model showing the impact of the arginine to proline substitution at position 1667. The mutant stick structure (blue) is overlaid on the wild-type stick structure (white) with R1667 shown in green and the R1667P substitution shown in red. In both cases, F1609 is shown; this residue is colored green and red in the in the wild-type and mutant structures, respectively.

Journal: Scientific Reports

Article Title: Complex effects on Ca V 2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder

doi: 10.1038/s41598-022-12789-y

Figure Lengend Snippet: The R1667P mutation occurs at the R4 position of the Repeat IV S4 voltage-sensing α-helix. ( a ) Schematic representation of Ca V 2.1 with Green Fluorescent Protein (GFP) fused to the amino-terminus (GFP-Ca V 2.1). The R to P substitution at residue 1667 is indicated by the red star. ( b ) Sequence comparison of the Repeat IV S4 helices of all known human Ca V 2.1 variants ( cf . accession no. NP_001120693.1), human Ca V 2.1 with the R to P substitution at the R4 position and human Ca V 2.2 (accession no. NM_001243812). Basic residues in positions R2-R6, as defined by Ca V 2.2 Cryo-EM structure are shown in green and the R4 R to P substitution is shown in red. ( c , d ) AlphaFold2 modeling of the voltage-sensing module of Repeat IV of human Ca V 2.1 . The S1–S4 helices are viewed from the lateral aspect ( c ) and from an extracellular ( d ) vantage points. Potential hydrogen bonds between R1667 and N1579 in S1, T1606 in S2, and S1641 in S3 are indicated by the blue dashed lines. F1609 (i.e., the gating charge transfer center) is also labelled. Panels ( c ) and ( d ) were published with permission https://creativecommons.org/licenses/by/4.0/ ( e ) Missense 3D model showing the impact of the arginine to proline substitution at position 1667. The mutant stick structure (blue) is overlaid on the wild-type stick structure (white) with R1667 shown in green and the R1667P substitution shown in red. In both cases, F1609 is shown; this residue is colored green and red in the in the wild-type and mutant structures, respectively.

Article Snippet: To generate GFP-Ca V 2.1 R1667P, a guanine to cytosine substitution at bp 5000 of the sequence encoding Ca V 2.1 (Δ10A (− V + G), 16 + 17 + , Δ17A (− VEA), − 31* (− NP), 37a (EFa), 43 + 44 + , Δ47) was introduced into the plasmid by Genscript, Inc. using proprietary methods.

Techniques: Mutagenesis, Residue, Sequencing, Comparison, Cryo-EM Sample Prep

The R1667P mutation causes a profound reduction in Ca 2+ current density and a hyperpolarizing shift in Ca V 2.1 activation. ( a ) Ca 2+ current families recorded from tsA-201 cells expressing GFP-Ca V 2.1 ( left ) or GFP-Ca V 2.1 R1667P ( right ) with auxiliary β 4 and α 2 δ-1 subunits. Currents were elicited by a 25 ms step depolarizations from − 80 mV to indicated test potentials; the repolarization voltage was − 40 mV. Confocal images confirming successful heterologous expression of GFP-Ca V 2.1 and GFP-Ca V 2.1 R1667P are shown in the insets . Scale bars = 10 µm. ( b ) Comparison of GFP-Ca V 2.1 (filled circle; n = 17) and GFP-Ca V 2.1 R1667P (open circle; n = 19) average peak I–V relationships. Currents were evoked at 0.1 Hz by test potentials ranging from − 50 mV through + 80 mV in 10 mV increments. Amplitudes were normalized by capacitance (pA/pF). ( c ) Normalized G–V curves were fit by Eq. with the following respective parameters for GFP-Ca V 2.1 and GFP-Ca V 2.1 R1667P: V G = 0.9 ± 0.7 and − 10.0 ± 0.9 mV; k = 5.4 ± 0.2 and 6.6 ± 0.8 mV, respectively. Throughout, data are presented as mean ± SEM; the total number of cells in a data set is indicated in parentheses.

Journal: Scientific Reports

Article Title: Complex effects on Ca V 2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder

doi: 10.1038/s41598-022-12789-y

Figure Lengend Snippet: The R1667P mutation causes a profound reduction in Ca 2+ current density and a hyperpolarizing shift in Ca V 2.1 activation. ( a ) Ca 2+ current families recorded from tsA-201 cells expressing GFP-Ca V 2.1 ( left ) or GFP-Ca V 2.1 R1667P ( right ) with auxiliary β 4 and α 2 δ-1 subunits. Currents were elicited by a 25 ms step depolarizations from − 80 mV to indicated test potentials; the repolarization voltage was − 40 mV. Confocal images confirming successful heterologous expression of GFP-Ca V 2.1 and GFP-Ca V 2.1 R1667P are shown in the insets . Scale bars = 10 µm. ( b ) Comparison of GFP-Ca V 2.1 (filled circle; n = 17) and GFP-Ca V 2.1 R1667P (open circle; n = 19) average peak I–V relationships. Currents were evoked at 0.1 Hz by test potentials ranging from − 50 mV through + 80 mV in 10 mV increments. Amplitudes were normalized by capacitance (pA/pF). ( c ) Normalized G–V curves were fit by Eq. with the following respective parameters for GFP-Ca V 2.1 and GFP-Ca V 2.1 R1667P: V G = 0.9 ± 0.7 and − 10.0 ± 0.9 mV; k = 5.4 ± 0.2 and 6.6 ± 0.8 mV, respectively. Throughout, data are presented as mean ± SEM; the total number of cells in a data set is indicated in parentheses.

Techniques: Mutagenesis, Activation Assay, Expressing, Comparison

The R1667P mutation slows activation and deactivation. Ca 2+ currents were recorded from tsA-201 cells expressing either GFP-Ca V 2.1 ( a ) or GFP-Ca V 2.1 R1667P ( b ). Currents were elicited by 25 ms step depolarizations from − 80 mV to test potentials ranging from − 20 mV through + 30 mV ( a - top ). Activation was fit by Eq. ; the time constants of activation (τ act ) for representative cells are indicated. ( c ) Comparison of τ act for GFP-Ca V 2.1 (filled circle; n = 17) or GFP-Ca V 2.1 R1667P (open circle; n = 15) measured at the indicated test potentials. Representative tail currents were recorded from tsA-201 cells expressing either GFP-Ca V 2.1 ( d ) or GFP-Ca V 2.1 R1667P ( e ) upon repolarization from + 30 mV to the indicated potentials ( d -top). Deactivation was fit by Eq. . ( f ) Comparison of τ deact for GFP-Ca V 2.1 (filled circle; n = 13) and GFP-Ca V 2.1 R1667P (open circle; n = 9) measured at the indicated repolarization potentials. Significant differences by two-tailed, unpaired t-test are indicated (*Denotes P < 0.05; ***Denotes P < 0.001).

Journal: Scientific Reports

Article Title: Complex effects on Ca V 2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder

doi: 10.1038/s41598-022-12789-y

Figure Lengend Snippet: The R1667P mutation slows activation and deactivation. Ca 2+ currents were recorded from tsA-201 cells expressing either GFP-Ca V 2.1 ( a ) or GFP-Ca V 2.1 R1667P ( b ). Currents were elicited by 25 ms step depolarizations from − 80 mV to test potentials ranging from − 20 mV through + 30 mV ( a - top ). Activation was fit by Eq. ; the time constants of activation (τ act ) for representative cells are indicated. ( c ) Comparison of τ act for GFP-Ca V 2.1 (filled circle; n = 17) or GFP-Ca V 2.1 R1667P (open circle; n = 15) measured at the indicated test potentials. Representative tail currents were recorded from tsA-201 cells expressing either GFP-Ca V 2.1 ( d ) or GFP-Ca V 2.1 R1667P ( e ) upon repolarization from + 30 mV to the indicated potentials ( d -top). Deactivation was fit by Eq. . ( f ) Comparison of τ deact for GFP-Ca V 2.1 (filled circle; n = 13) and GFP-Ca V 2.1 R1667P (open circle; n = 9) measured at the indicated repolarization potentials. Significant differences by two-tailed, unpaired t-test are indicated (*Denotes P < 0.05; ***Denotes P < 0.001).

Techniques: Mutagenesis, Activation Assay, Expressing, Comparison, Two Tailed Test

The R1667P mutation has little effect on closed-state inactivation. ( a ) A 5 s conditioning step from the steady holding potential (− 80 mV) to increasing potentials ranging from − 100 to + 30 mV (in 10 mV increments) was applied before repolarizing the membrane to − 80 mV for 5 ms. Test currents were then evoked by a 25 ms step depolarization to + 20 mV. The protocol is not drawn to scale. Representative Ca 2+ currents recorded from tsA-201 cells expressing GFP-Ca V 2.1 ( b ) or GFP-Ca V 2.1 R1667P ( c ) after pre-pulses to − 100, − 80, − 60, − 40, − 20, 0 and + 20 mV. Normalized steady-state inactivation curves for GFP-Ca V 2.1 (filled circle; n = 13) and GFP-Ca V 2.1 R1667P (open circle; n = 6) are shown in ( d ); amplitudes were normalized by the maximal Ca 2+ current in each cell. The normalized inactivation relationships were fit with Eq. with the following fit parameters for GFP-Ca V 2.1 and GFP-Ca V 2.1 R1667P: V 1/2inact = − 39.3 ± 15 and − 42.1 ± 1.8 mV; k = − 12.6 ± 0.6 and − 10.7 ± 0.7 mV, respectively. ( e ) Overlay of the smooth conductance and closed-state inactivation curves (from Figs. c and 4d, respectively) for cells expressing GFP-Ca V 2.1 (black lines) and GFP-Ca V 2.1 R1667P (red lines).

Journal: Scientific Reports

Article Title: Complex effects on Ca V 2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder

doi: 10.1038/s41598-022-12789-y

Figure Lengend Snippet: The R1667P mutation has little effect on closed-state inactivation. ( a ) A 5 s conditioning step from the steady holding potential (− 80 mV) to increasing potentials ranging from − 100 to + 30 mV (in 10 mV increments) was applied before repolarizing the membrane to − 80 mV for 5 ms. Test currents were then evoked by a 25 ms step depolarization to + 20 mV. The protocol is not drawn to scale. Representative Ca 2+ currents recorded from tsA-201 cells expressing GFP-Ca V 2.1 ( b ) or GFP-Ca V 2.1 R1667P ( c ) after pre-pulses to − 100, − 80, − 60, − 40, − 20, 0 and + 20 mV. Normalized steady-state inactivation curves for GFP-Ca V 2.1 (filled circle; n = 13) and GFP-Ca V 2.1 R1667P (open circle; n = 6) are shown in ( d ); amplitudes were normalized by the maximal Ca 2+ current in each cell. The normalized inactivation relationships were fit with Eq. with the following fit parameters for GFP-Ca V 2.1 and GFP-Ca V 2.1 R1667P: V 1/2inact = − 39.3 ± 15 and − 42.1 ± 1.8 mV; k = − 12.6 ± 0.6 and − 10.7 ± 0.7 mV, respectively. ( e ) Overlay of the smooth conductance and closed-state inactivation curves (from Figs. c and 4d, respectively) for cells expressing GFP-Ca V 2.1 (black lines) and GFP-Ca V 2.1 R1667P (red lines).

Techniques: Mutagenesis, Membrane, Expressing

The R1667P mutation reduces total Ca 2+ flux in response to a single action potential-like stimulus. Ca 2+ currents attributable to GFP-Ca V 2.1 ( a ) and GFP-Ca V 2.1 R1667P ( b ) were evoked by an action potential-like waveform consisting of a 1 ms ramp from − 80 mV to + 30 mV followed immediately by a 1 ms ramp back to − 80 mV. Traces shown are the average of 10 recordings. ( c ) Comparison of total charge flux normalized to cell membrane capacitance (fC/pF) for cells expressing GFP-Ca V 2.1 (filled circle; n = 13) or GFP-Ca V 2.1 R1667P (open circle; n = 12). ( d ) Absolute charge flux normalized to tail current amplitude at the reversal potential (i.e., maximal conductance) (fC/pA). Means are indicated by the dashed lines of the boxes. Boxes represent the 25th/75th percentiles. Bars represent the 5th/95th percentiles. A significant difference is indicated (***) in ( c ).

Journal: Scientific Reports

Article Title: Complex effects on Ca V 2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder

doi: 10.1038/s41598-022-12789-y

Figure Lengend Snippet: The R1667P mutation reduces total Ca 2+ flux in response to a single action potential-like stimulus. Ca 2+ currents attributable to GFP-Ca V 2.1 ( a ) and GFP-Ca V 2.1 R1667P ( b ) were evoked by an action potential-like waveform consisting of a 1 ms ramp from − 80 mV to + 30 mV followed immediately by a 1 ms ramp back to − 80 mV. Traces shown are the average of 10 recordings. ( c ) Comparison of total charge flux normalized to cell membrane capacitance (fC/pF) for cells expressing GFP-Ca V 2.1 (filled circle; n = 13) or GFP-Ca V 2.1 R1667P (open circle; n = 12). ( d ) Absolute charge flux normalized to tail current amplitude at the reversal potential (i.e., maximal conductance) (fC/pA). Means are indicated by the dashed lines of the boxes. Boxes represent the 25th/75th percentiles. Bars represent the 5th/95th percentiles. A significant difference is indicated (***) in ( c ).

Techniques: Mutagenesis, Comparison, Membrane, Expressing

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Inhibition of Cav2.1 Ca2+ currents by the R1279X EA2-truncated form of Cav2.1 protein. A, Schematic cartoon representing the main Cav2.1 constructs used in this study. B, The Ca2+ current density in HEK293 cells expressing the Cav2.1 channel protein. The cells were cotransfected with plasmids encoding the human Cav2.1 subunit, the β1b subunit, and the α2/δ1 subunit. Mean ± SEM values of the current density for cells expressing the Cav2.1 subunit alone (white bar; n = 18) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22) are presented (***p < 0.001; Student's t test). The inset shows representative current traces at TP −40 and 0 mV [holding potential (HP), −80 mV]. C, Same experiments as in B performed in the neuroblastoma cell line NG108-15. The HP was −50 mV (***p < 0.001; Student's t test). Cav2.1 subunit alone (white bar; n = 15) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22). D, Measurements of native T-type and HVA Ca2+ current densities in R1279X-transfected NG108-15 cells. The T-current density (left) was measured in proliferative NG108-15 cells (Prolif.; 2–3 d after transfection; n = 9) and in differentiated NG108-15 cells (Diff.; 6 d after transfection; 3–4 d after switching to differentiation medium; n = 11) using HP −100 mV and TP −30 mV. The HVA current density, mainly corresponding to L- and N-type channel activities, was measured using HP −50 mV and TP 0 mV (right).

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Inhibition, Construct, Expressing, Mutagenesis, Transfection

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Effect of R1279X mutant on surface expression of Cav2.1 channel. A, HEK293 cells were cotransfected with the HA-tagged wild-type Cav2.1 subunit alone (with free GFP) or together with the R1279X mutant (fused to GFP). Left column, GFP fluorescence. Middle column, Staining of cells with monoclonal rat anti-HA antibody (primary antibody) and Alexa594 (secondary antibody). NP, Nonpermeabilized; P, permeabilized. Images were acquired using a Leica SP2 confocal microscope, with a 63× oil immersion objective. B, Luminometric ELISA assays to quantify surface expression of the HA-tagged Cav2.1 in the presence of the R1279X mutant and auxiliary subunits (**p < 0.01, ***p < 0.001; Student's t test). C, Western blots performed on HEK293 cells expressing Cav2.1-HA alone (−) or in the presence of R1279X (+) without (left) or with (right) auxiliary subunits. β1b-HA was used in these experiments (right).

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Mutagenesis, Expressing, Fluorescence, Staining, Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Channel misfolding and instability induced by the R1279X mutant. A, B, Pulse-chase experiments were performed on HEK293 cells 48 h after transfection (see Materials and Methods). Cells were labeled with 35S-methionine-cysteine for 20 min. The chase was performed for 0, 1, 2, or 4 h as indicated. After lysis, the Cav2.1 subunit was immunoprecipitated with an anti-HA antibody. The arrow indicates the band corresponding to the EA2 mutant that coimmunoprecipitates with the wild-type Cav2.1 subunit. In B (left), the β1b is detected (arrow). C, Quantification of three independent experiments using GE Healthcare software. D, Pulse-chase analyses performed on HEK293 cells transfected with CD4-AAXX alone or in the presence of R1279X. Quantification was performed as described above. E, Immunoprecipitations performed on cells coexpressing various subunit arrangements (as indicated) in the absence or presence of an untagged R1279X mutant (right) using standard SDS-PAGE (6%). Western blots (WBs) were performed with the indicated antibodies. F, Immunoprecipitations were performed between β1b-HA and GFP-R1279X as described in E. MW, Molecular weight.

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Mutagenesis, Pulse Chase, Transfection, Labeling, Lysis, Immunoprecipitation, Software, SDS Page, Western Blot, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: EA2 missense mutants act in a dominant-negative manner by misfolding and instability induction. A, Histograms of the mean Ca2+ current density (± SEM) obtained in a representative batch of NG108-15 cells expressing wild-type subunit alone (Cav2.1-HA, n = 18); the EA2 mutants alone (Cav2.1-HA-G293R, n = 33, 5 with detectable current; Cav2.1-HA-AY1593/94D, n = 6); and the wild-type and EA2 mutants together (Cav2.1-HA+Cav2.1-HA-G293R, n = 23, 3 with detectable current; Cav2.1-HA+Cav2.1-HA-AY1593/94D, n = 10). These experiments were conducted in the presence of the auxiliary β1b and α2/δ1 subunits. B, Normalized native T-type Ca2+ current densities (mean ± SEM) in the NG108-15 cells analyzed in A. The differences are not statistically significant between the various conditions tested (Student's t test). C, Western blots with the indicated antibodies (WB) were performed on HEK293 cells coexpressing Cav2.1-HA alone or in the presence of G293R and AY1593/94D mutants together with auxiliary subunits. The bottom panel shows the Western blot from cells expressing missense mutants alone. D, Pulse-chase analyses were performed on HEK293 cells transfected with wild-type Cav2.1 alone or in the presence of G293R. Quantification of three independents experiment was performed as described above. MW, Molecular weight.

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Dominant Negative Mutation, Expressing, Western Blot, Pulse Chase, Transfection, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Endoplasmic reticulum retention and proteasomal degradation of the dominant-negative Cav mutants. A, Confocal images of nonpermeabilized NG108-15 cells expressing EA2 mutants and truncated Cav3.2 subunits. Alexa 594-coupled CT was used as plasma membrane marker (0.5 μg/ml). B, Confocal images of immunofluorescence staining performed on permeabilized NG108-15 cells expressing indicated EA2 mutants and truncated Cav3.2. Polyclonal anti-protein disulfide isomerase (Assay Designs, Ann Arbor, MI) was used as ER marker. C, Pulse-chase experiments were performed as in Figure 3 on cells transfected with EA2 mutants (R1279X and G293R) and Cav3.2 truncated forms. Chase was done for the indicated time (hours) with or without MG-132 proteasome inhibitor (50 μm). After lysis, the truncated Cav channels were immunoprecipitated with anti-GFP antibody. D, Representative pulse chase performed on HEK293 cells transfected with Cav2.1 and the R1279X mutant and the corresponding quantification (n = 3). During the chase, cells were treated with MG-132 (50 μm), leupeptin (20 μm), and NH4Cl (10 mm).

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Dominant Negative Mutation, Expressing, Marker, Immunofluorescence, Staining, Pulse Chase, Transfection, Lysis, Immunoprecipitation, Mutagenesis

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