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Disruption of the IS6-AID linker affects voltage-gated calcium channel inactivation and facilitation.

Findeisen F, Minor DL - J. Gen. Physiol. (2009)

Bottom Line: The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively.Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved.Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, Department of Biochemistry and Biophysics, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, USA.

ABSTRACT
Two processes dominate voltage-gated calcium channel (Ca(V)) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6-alpha-interaction domain (AID) linker provides a rigid connection between the pore and Ca(V)beta/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate Ca(V)1.2 (L-type) and Ca(V)2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt Ca(V)beta/I-II association sharply decelerate CDI and reduce a second Ca(2+)/CaM/Ca(V)alpha(1)-C-terminal-mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.

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Glycine substitution in the IS6-AID linker affects VDI. (A) Amino acid sequence of wild-type and mutant IS6-AID linker sequences from CaV1.2 and CaV2.1. SOPMA secondary structure prediction is indicated (Geourjon and Deleage, 1995). (B) Disruption of the IS6-AID linker accelerates CaV1.2 VDI. Representative normalized IBa traces at a test potential of +20 mV for the combination of the indicated CaV1.2 subunits and CaVβ2a. (C) ti300 values for data from B. Results of unpaired t tests or one-way ANOVA, as appropriate, are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (D) G-V relationships in barium for the indicated combinations of CaV1.2 subunits and CaVβ2a. (E) Disruption of the IS6-AID linker accelerates CaV2.1 VDI. Representative normalized IBa traces of CaV2.1 wild-type and CaV2.1 GGG. Data in all figures are represented as mean ± SD.
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fig1: Glycine substitution in the IS6-AID linker affects VDI. (A) Amino acid sequence of wild-type and mutant IS6-AID linker sequences from CaV1.2 and CaV2.1. SOPMA secondary structure prediction is indicated (Geourjon and Deleage, 1995). (B) Disruption of the IS6-AID linker accelerates CaV1.2 VDI. Representative normalized IBa traces at a test potential of +20 mV for the combination of the indicated CaV1.2 subunits and CaVβ2a. (C) ti300 values for data from B. Results of unpaired t tests or one-way ANOVA, as appropriate, are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (D) G-V relationships in barium for the indicated combinations of CaV1.2 subunits and CaVβ2a. (E) Disruption of the IS6-AID linker accelerates CaV2.1 VDI. Representative normalized IBa traces of CaV2.1 wild-type and CaV2.1 GGG. Data in all figures are represented as mean ± SD.

Mentions: As noted previously (Opatowsky et al., 2004; Van Petegem et al., 2004), the IS6-AID linker has a high probability to form an α-helical structure (Fig. 1 A). To disrupt the integrity of this putative helix, we mutated three consecutive residues in the middle of the CaV1.2 IS6-AID linker, residues 415–417, to glycine (denoted as GGG) (Fig. 1 A). Due to the extremely low helix propensity of glycine (O’Neil and DeGrado, 1990; Blaber et al., 1993), the GGG mutation is expected to cause substantial disruption of any helical structure present in the IS6-AID linker, as it should incur an ∼3–kcal mol−1 destabilization of the helical conformation. As a control for effects resulting from side chain deletion, we also made a mutant that converts CaV1.2 residues 415–417 into a triple-alanine sequence, denoted as AAA. Based on the high helix–forming propensity of alanine, this substitution should leave the IS6-AID helix intact.


Disruption of the IS6-AID linker affects voltage-gated calcium channel inactivation and facilitation.

Findeisen F, Minor DL - J. Gen. Physiol. (2009)

Glycine substitution in the IS6-AID linker affects VDI. (A) Amino acid sequence of wild-type and mutant IS6-AID linker sequences from CaV1.2 and CaV2.1. SOPMA secondary structure prediction is indicated (Geourjon and Deleage, 1995). (B) Disruption of the IS6-AID linker accelerates CaV1.2 VDI. Representative normalized IBa traces at a test potential of +20 mV for the combination of the indicated CaV1.2 subunits and CaVβ2a. (C) ti300 values for data from B. Results of unpaired t tests or one-way ANOVA, as appropriate, are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (D) G-V relationships in barium for the indicated combinations of CaV1.2 subunits and CaVβ2a. (E) Disruption of the IS6-AID linker accelerates CaV2.1 VDI. Representative normalized IBa traces of CaV2.1 wild-type and CaV2.1 GGG. Data in all figures are represented as mean ± SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654080&req=5

fig1: Glycine substitution in the IS6-AID linker affects VDI. (A) Amino acid sequence of wild-type and mutant IS6-AID linker sequences from CaV1.2 and CaV2.1. SOPMA secondary structure prediction is indicated (Geourjon and Deleage, 1995). (B) Disruption of the IS6-AID linker accelerates CaV1.2 VDI. Representative normalized IBa traces at a test potential of +20 mV for the combination of the indicated CaV1.2 subunits and CaVβ2a. (C) ti300 values for data from B. Results of unpaired t tests or one-way ANOVA, as appropriate, are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (D) G-V relationships in barium for the indicated combinations of CaV1.2 subunits and CaVβ2a. (E) Disruption of the IS6-AID linker accelerates CaV2.1 VDI. Representative normalized IBa traces of CaV2.1 wild-type and CaV2.1 GGG. Data in all figures are represented as mean ± SD.
Mentions: As noted previously (Opatowsky et al., 2004; Van Petegem et al., 2004), the IS6-AID linker has a high probability to form an α-helical structure (Fig. 1 A). To disrupt the integrity of this putative helix, we mutated three consecutive residues in the middle of the CaV1.2 IS6-AID linker, residues 415–417, to glycine (denoted as GGG) (Fig. 1 A). Due to the extremely low helix propensity of glycine (O’Neil and DeGrado, 1990; Blaber et al., 1993), the GGG mutation is expected to cause substantial disruption of any helical structure present in the IS6-AID linker, as it should incur an ∼3–kcal mol−1 destabilization of the helical conformation. As a control for effects resulting from side chain deletion, we also made a mutant that converts CaV1.2 residues 415–417 into a triple-alanine sequence, denoted as AAA. Based on the high helix–forming propensity of alanine, this substitution should leave the IS6-AID helix intact.

Bottom Line: The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively.Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved.Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, Department of Biochemistry and Biophysics, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, USA.

ABSTRACT
Two processes dominate voltage-gated calcium channel (Ca(V)) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6-alpha-interaction domain (AID) linker provides a rigid connection between the pore and Ca(V)beta/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate Ca(V)1.2 (L-type) and Ca(V)2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt Ca(V)beta/I-II association sharply decelerate CDI and reduce a second Ca(2+)/CaM/Ca(V)alpha(1)-C-terminal-mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.

Show MeSH
Related in: MedlinePlus