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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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IS6-AID linker disruption reduces CDI. (A) Representative netCDI (ICa/IBa) at a test potential of +20 mV for the combinations of the indicated CaV1.2 subunits and CaVβ2a. (B) ti300 values from A. Results of unpaired t tests are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (C) Isochronal inactivation of CaV1.2 (n = 4, black X’s, netCDI; n = 4, black open squares, VDI), CaV1.2 GGG netCDI (n = 4, gray), and CaV1.2 GGG/HotA netCDI (n = 5, orange). Inactivation extent comparing the ratio of prepulse and test pulse current amplitudes plotted as a function of the test voltage. The pulse protocol is shown at the top. (D) G-V relationships in calcium for the indicated combinations of CaV1.2 subunits and CaVβ2a.
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fig4: IS6-AID linker disruption reduces CDI. (A) Representative netCDI (ICa/IBa) at a test potential of +20 mV for the combinations of the indicated CaV1.2 subunits and CaVβ2a. (B) ti300 values from A. Results of unpaired t tests are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (C) Isochronal inactivation of CaV1.2 (n = 4, black X’s, netCDI; n = 4, black open squares, VDI), CaV1.2 GGG netCDI (n = 4, gray), and CaV1.2 GGG/HotA netCDI (n = 5, orange). Inactivation extent comparing the ratio of prepulse and test pulse current amplitudes plotted as a function of the test voltage. The pulse protocol is shown at the top. (D) G-V relationships in calcium for the indicated combinations of CaV1.2 subunits and CaVβ2a.

Mentions: A previous study of wild-type CaV1.2 channels expressed in a mammalian cell line suggested that in some cases, IBa may also undergo current-dependent inactivation (Ferreira et al., 1997), a condition that if present to a substantial degree would complicate the netCDI analysis. To test whether such a phenomenon occurred in our Xenopus oocyte experimental setup, we examined the inactivation properties of IBa using a double-pulse protocol (Fig. 4 C). Comparison of the IBa prepulse and test pulse amplitudes does not reveal a U-shaped dependence that would be a signature of VDI current-dependent inactivation (Fig. 4 C) and supports the use of netCDI as a means for parsing inactivation into effects from CDI and VDI.


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

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

IS6-AID linker disruption reduces CDI. (A) Representative netCDI (ICa/IBa) at a test potential of +20 mV for the combinations of the indicated CaV1.2 subunits and CaVβ2a. (B) ti300 values from A. Results of unpaired t tests are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (C) Isochronal inactivation of CaV1.2 (n = 4, black X’s, netCDI; n = 4, black open squares, VDI), CaV1.2 GGG netCDI (n = 4, gray), and CaV1.2 GGG/HotA netCDI (n = 5, orange). Inactivation extent comparing the ratio of prepulse and test pulse current amplitudes plotted as a function of the test voltage. The pulse protocol is shown at the top. (D) G-V relationships in calcium for the indicated combinations of CaV1.2 subunits and CaVβ2a.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: IS6-AID linker disruption reduces CDI. (A) Representative netCDI (ICa/IBa) at a test potential of +20 mV for the combinations of the indicated CaV1.2 subunits and CaVβ2a. (B) ti300 values from A. Results of unpaired t tests are indicated as follows: N.S., P > 0.05, not significant; ***, P < 0.001. (C) Isochronal inactivation of CaV1.2 (n = 4, black X’s, netCDI; n = 4, black open squares, VDI), CaV1.2 GGG netCDI (n = 4, gray), and CaV1.2 GGG/HotA netCDI (n = 5, orange). Inactivation extent comparing the ratio of prepulse and test pulse current amplitudes plotted as a function of the test voltage. The pulse protocol is shown at the top. (D) G-V relationships in calcium for the indicated combinations of CaV1.2 subunits and CaVβ2a.
Mentions: A previous study of wild-type CaV1.2 channels expressed in a mammalian cell line suggested that in some cases, IBa may also undergo current-dependent inactivation (Ferreira et al., 1997), a condition that if present to a substantial degree would complicate the netCDI analysis. To test whether such a phenomenon occurred in our Xenopus oocyte experimental setup, we examined the inactivation properties of IBa using a double-pulse protocol (Fig. 4 C). Comparison of the IBa prepulse and test pulse amplitudes does not reveal a U-shaped dependence that would be a signature of VDI current-dependent inactivation (Fig. 4 C) and supports the use of netCDI as a means for parsing inactivation into effects from CDI and VDI.

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