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A Ca V 2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant

View Article: PubMed Central - PubMed

ABSTRACT

Episodic ataxia 2 (EA2) is an autosomal dominant disorder caused by mutations in the gene CACNA1A that encodes the pore-forming CaV2.1 calcium channel subunit. The majority of EA2 mutations reported so far are nonsense or deletion/insertion mutations predicted to form truncated proteins. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide.

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Rescue by the free N-terminus of the suppression by EA2 of endogenous P/Q type current in DRG neurons.(A) Confocal images of DRG neurons transfected with empty pcDNA3 vector and YFP (control, top panel), EA2, pcDNA3 empty vector and YFP (middle panel) or EA2, CaV2.1-(46-100)-CAAX and YFP (bottom panel). The neurons were permeabilized and stained with CaV2.1 antibody that targets the II–III loop. (B) Representative traces of endogenous P/Q current. The DRG neurons were co-transfected with empty pcDNA3 vector and YFP (black); EA2 and pcDNA3 empty vector and YFP (red) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue). The P/Q-type current was isolated pharmacologically. Currents were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV. The charge carrier was 5 mM Ba2 +. (C) Current-voltage relationships of DRG neurons transfected with pcDNA3 empty vector and YFP (black squares, n = 13), EA2, pcDNA3 empty vector and YFP (red circles, n = 10) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue circles, n = 15). (D) Current density at + 10 mV ± SEM for pcDNA3 empty vector and YFP (black, n = 13), EA2, pcDNA3 empty vector and YFP (red, n = 10) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue, n = 15). Statistical analysis: *p < 0.05, **p < 0.01, ns = non-significant difference.
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f0040: Rescue by the free N-terminus of the suppression by EA2 of endogenous P/Q type current in DRG neurons.(A) Confocal images of DRG neurons transfected with empty pcDNA3 vector and YFP (control, top panel), EA2, pcDNA3 empty vector and YFP (middle panel) or EA2, CaV2.1-(46-100)-CAAX and YFP (bottom panel). The neurons were permeabilized and stained with CaV2.1 antibody that targets the II–III loop. (B) Representative traces of endogenous P/Q current. The DRG neurons were co-transfected with empty pcDNA3 vector and YFP (black); EA2 and pcDNA3 empty vector and YFP (red) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue). The P/Q-type current was isolated pharmacologically. Currents were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV. The charge carrier was 5 mM Ba2 +. (C) Current-voltage relationships of DRG neurons transfected with pcDNA3 empty vector and YFP (black squares, n = 13), EA2, pcDNA3 empty vector and YFP (red circles, n = 10) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue circles, n = 15). (D) Current density at + 10 mV ± SEM for pcDNA3 empty vector and YFP (black, n = 13), EA2, pcDNA3 empty vector and YFP (red, n = 10) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue, n = 15). Statistical analysis: *p < 0.05, **p < 0.01, ns = non-significant difference.

Mentions: We then examined whether CaV2.1-(46-100)-CAAX could restore the endogenous P/Q-type current in DRG neurons co-transfected with the EA2 mutant. Firstly, DRG neurons were transfected with YFP as a transfection marker and empty vector, EA2 or EA2 plus CaV2.1-(46-100)-CAAX, and stained with a CaV2.1 antibody that targets the II–III loop present in EA2, to demonstrate its expression (Fig. 8A). The native CaV2.1 could not be detected, probably because of its low level of expression. We performed experiments 4 days after transfection in the presence of 1 μM nifedipine and 1 μM ω-conotoxin GVIA to block L- and N-type channels, respectively, in order to isolate native P/Q-type calcium currents. The electrophysiological data showed that the expression of EA2 mutant induced, as expected, a reduction of the native P/Q-type current greater than 50% (control: − 66.0 ± 10.9 pA/pF; EA2: − 27.6 ± 4.3 pA/pF; Fig. 8B–D). Importantly, this reduction was almost completely prevented when CaV2.1-(46-100)-CAAX was co-expressed (− 65.0 ± 8.8 pA/pF; Fig. 8B–D). This finding further reinforces the view that key N-terminal residues interfere with the dominant-negative effect of EA2.


A Ca V 2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant
Rescue by the free N-terminus of the suppression by EA2 of endogenous P/Q type current in DRG neurons.(A) Confocal images of DRG neurons transfected with empty pcDNA3 vector and YFP (control, top panel), EA2, pcDNA3 empty vector and YFP (middle panel) or EA2, CaV2.1-(46-100)-CAAX and YFP (bottom panel). The neurons were permeabilized and stained with CaV2.1 antibody that targets the II–III loop. (B) Representative traces of endogenous P/Q current. The DRG neurons were co-transfected with empty pcDNA3 vector and YFP (black); EA2 and pcDNA3 empty vector and YFP (red) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue). The P/Q-type current was isolated pharmacologically. Currents were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV. The charge carrier was 5 mM Ba2 +. (C) Current-voltage relationships of DRG neurons transfected with pcDNA3 empty vector and YFP (black squares, n = 13), EA2, pcDNA3 empty vector and YFP (red circles, n = 10) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue circles, n = 15). (D) Current density at + 10 mV ± SEM for pcDNA3 empty vector and YFP (black, n = 13), EA2, pcDNA3 empty vector and YFP (red, n = 10) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue, n = 15). Statistical analysis: *p < 0.05, **p < 0.01, ns = non-significant difference.
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f0040: Rescue by the free N-terminus of the suppression by EA2 of endogenous P/Q type current in DRG neurons.(A) Confocal images of DRG neurons transfected with empty pcDNA3 vector and YFP (control, top panel), EA2, pcDNA3 empty vector and YFP (middle panel) or EA2, CaV2.1-(46-100)-CAAX and YFP (bottom panel). The neurons were permeabilized and stained with CaV2.1 antibody that targets the II–III loop. (B) Representative traces of endogenous P/Q current. The DRG neurons were co-transfected with empty pcDNA3 vector and YFP (black); EA2 and pcDNA3 empty vector and YFP (red) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue). The P/Q-type current was isolated pharmacologically. Currents were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV. The charge carrier was 5 mM Ba2 +. (C) Current-voltage relationships of DRG neurons transfected with pcDNA3 empty vector and YFP (black squares, n = 13), EA2, pcDNA3 empty vector and YFP (red circles, n = 10) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue circles, n = 15). (D) Current density at + 10 mV ± SEM for pcDNA3 empty vector and YFP (black, n = 13), EA2, pcDNA3 empty vector and YFP (red, n = 10) or EA2, CaV2.1-(46-100)-CAAX and YFP (blue, n = 15). Statistical analysis: *p < 0.05, **p < 0.01, ns = non-significant difference.
Mentions: We then examined whether CaV2.1-(46-100)-CAAX could restore the endogenous P/Q-type current in DRG neurons co-transfected with the EA2 mutant. Firstly, DRG neurons were transfected with YFP as a transfection marker and empty vector, EA2 or EA2 plus CaV2.1-(46-100)-CAAX, and stained with a CaV2.1 antibody that targets the II–III loop present in EA2, to demonstrate its expression (Fig. 8A). The native CaV2.1 could not be detected, probably because of its low level of expression. We performed experiments 4 days after transfection in the presence of 1 μM nifedipine and 1 μM ω-conotoxin GVIA to block L- and N-type channels, respectively, in order to isolate native P/Q-type calcium currents. The electrophysiological data showed that the expression of EA2 mutant induced, as expected, a reduction of the native P/Q-type current greater than 50% (control: − 66.0 ± 10.9 pA/pF; EA2: − 27.6 ± 4.3 pA/pF; Fig. 8B–D). Importantly, this reduction was almost completely prevented when CaV2.1-(46-100)-CAAX was co-expressed (− 65.0 ± 8.8 pA/pF; Fig. 8B–D). This finding further reinforces the view that key N-terminal residues interfere with the dominant-negative effect of EA2.

View Article: PubMed Central - PubMed

ABSTRACT

Episodic ataxia 2 (EA2) is an autosomal dominant disorder caused by mutations in the gene CACNA1A that encodes the pore-forming CaV2.1 calcium channel subunit. The majority of EA2 mutations reported so far are nonsense or deletion/insertion mutations predicted to form truncated proteins. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide.

No MeSH data available.


Related in: MedlinePlus