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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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CDF is reduced by disruption of the IS6-AID linker and loss of CaVβ binding. (A) Relative current increase between the last (40th) and first +20-mV pulses at 3 Hz for CaV1.2 and the indicated mutants. Parentheses indicate the number of oocytes tested. Results of unpaired t tests are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (B) Exemplar current traces for CaV1.2 I1624A, CaV1.2 GGG/I1624A, and CaV1.2 HotA/I1624A in a 3-Hz 40-pulse train normalized to the peak of the first pulse.
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fig6: CDF is reduced by disruption of the IS6-AID linker and loss of CaVβ binding. (A) Relative current increase between the last (40th) and first +20-mV pulses at 3 Hz for CaV1.2 and the indicated mutants. Parentheses indicate the number of oocytes tested. Results of unpaired t tests are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (B) Exemplar current traces for CaV1.2 I1624A, CaV1.2 GGG/I1624A, and CaV1.2 HotA/I1624A in a 3-Hz 40-pulse train normalized to the peak of the first pulse.

Mentions: CaV1.2 channels display a phenomenon known as CDF, which is unmasked in the context of a point mutation, I1624A, in the C-terminal tail IQ domain that comprises the primary Ca2+/CaM binding site (Zühlke et al., 1999, 2000; Hudmon et al., 2005; Van Petegem et al., 2005). Given the pronounced effects of the IS6-AID linker GGG mutation and the importance of CaVβ for both VDI and CDI, we tested whether CDF might also rely upon an intact IS6-AID linker or the CaVβ subunit. Under conditions of similar current amplitude, the GGG mutation causes a reduction in CaV1.2 I1624A CDF of almost two thirds (13.8 ± 8.2% GGG/I1624A vs. 38.9 ± 12.3% I1624A, respectively). In contrast, the corresponding AAA mutant did not alter CDF significantly (37.5 ± 12.1%). CaV1.2 I1624A channels incapable of binding CaVβ through the incorporation of the HotA mutations show a complete absence of CDF (Fig. 6). This result agrees with the observation that CaVβ is central to the CDF mechanism (Grueter et al., 2006). Collectively, these data suggest that CDF requires both an intact IS6-AID linker and a CaVβ subunit, and that both CaV1.2 netCDI and CDF use the same components, including the IS6-AID linker and CaVβ for communicating with the transmembrane pore.


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

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

CDF is reduced by disruption of the IS6-AID linker and loss of CaVβ binding. (A) Relative current increase between the last (40th) and first +20-mV pulses at 3 Hz for CaV1.2 and the indicated mutants. Parentheses indicate the number of oocytes tested. Results of unpaired t tests are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (B) Exemplar current traces for CaV1.2 I1624A, CaV1.2 GGG/I1624A, and CaV1.2 HotA/I1624A in a 3-Hz 40-pulse train normalized to the peak of the first pulse.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: CDF is reduced by disruption of the IS6-AID linker and loss of CaVβ binding. (A) Relative current increase between the last (40th) and first +20-mV pulses at 3 Hz for CaV1.2 and the indicated mutants. Parentheses indicate the number of oocytes tested. Results of unpaired t tests are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (B) Exemplar current traces for CaV1.2 I1624A, CaV1.2 GGG/I1624A, and CaV1.2 HotA/I1624A in a 3-Hz 40-pulse train normalized to the peak of the first pulse.
Mentions: CaV1.2 channels display a phenomenon known as CDF, which is unmasked in the context of a point mutation, I1624A, in the C-terminal tail IQ domain that comprises the primary Ca2+/CaM binding site (Zühlke et al., 1999, 2000; Hudmon et al., 2005; Van Petegem et al., 2005). Given the pronounced effects of the IS6-AID linker GGG mutation and the importance of CaVβ for both VDI and CDI, we tested whether CDF might also rely upon an intact IS6-AID linker or the CaVβ subunit. Under conditions of similar current amplitude, the GGG mutation causes a reduction in CaV1.2 I1624A CDF of almost two thirds (13.8 ± 8.2% GGG/I1624A vs. 38.9 ± 12.3% I1624A, respectively). In contrast, the corresponding AAA mutant did not alter CDF significantly (37.5 ± 12.1%). CaV1.2 I1624A channels incapable of binding CaVβ through the incorporation of the HotA mutations show a complete absence of CDF (Fig. 6). This result agrees with the observation that CaVβ is central to the CDF mechanism (Grueter et al., 2006). Collectively, these data suggest that CDF requires both an intact IS6-AID linker and a CaVβ subunit, and that both CaV1.2 netCDI and CDF use the same components, including the IS6-AID linker and CaVβ for communicating with the transmembrane pore.

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