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J Gen Physiol
2009 Mar 01;1333:327-43. doi: 10.1085/jgp.200810143.
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Disruption of the IS6-AID linker affects voltage-gated calcium channel inactivation and facilitation.
Findeisen F
,
Minor DL
.
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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.
Figure 1. 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.
Figure 2. Glycine substitution in IS6-AID linker disrupts helical structure. (A) Mean residue ellipticity at 222 nm for IS6-AID linker peptide, and AAA and GGG mutant peptides as a function of TFE concentration. Peptide sequence is shown. Black highlights the site of the GGG and AAA mutations. (B) IS6-AID linker peptide CD spectra at a peptide concentration of 50 µM in 50% TFE.
Figure 3. CaVβ isoform rank order VDI effects remain in the presence of disrupted IS6-AID linker. Representative normalized IBa traces for the indicated CaVβ subunits coexpressed with (A) CaV1.2 and (B) CaV1.2 GGG. Note the different timescales. (C) Representative normalized IBa traces for coexpression of CaVβ1 and CaVβ2a with CaV2.1 and CaV2.1 GGG.
Figure 4. 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.
Figure 5. Effects of CaVβ isoforms on calcium inactivation stem from underlying effects on VDI. Normalized inactivation curves measured at +20 mV for (A) CaV1.2 and (B) CaV1.2 GGG subunits coexpressed with CaVβ1, CaVβ2a, CaVβ2b, or in the absence of CaVβ. (Left) VDI is shown and is reproduced for comparison from the first 300 ms of Fig. 3 (A and B). (Middle) Inactivation in calcium. (Right) netCDI.
Figure 6. 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.
Figure 7. Cartoon model of a CaV channel. Based on the likely gross similarly between CaV and Kv transmembrane portions, the Kv1.2 transmembrane domains (gray surface; PDB accession no. 2A79) are used to represent the CaV transmembrane domains. The IS6-AID linker (red) was modeled manually by building a helix of corresponding length between the Kv1.2 S6 helix C terminus (dark gray) and AID helix (light gray) from the CaVβ2a–AID complex (PDB accession no. 1T0J). The CaVβ2a–AID complex is shown as follows: green, SH3 domain; light blue, NK domain; light gray, AID. N-terminal CaVβ2a variable segment, V1, of unknown structure, is shown anchored to the membrane via N-terminal palmitoylation, and the V2 loop is indicated. Arrow along the IS6-AID linker indicates communication between CaVβ and the pore domain. This is lost in the multiple glycine mutants (bottom) and affects VDI, CDI, and CDF. Curved arrow between the Ca2+/CaM-IQ domain complex (PDB accession no. 2BE6), Ca2+/CaM (dark blue), and IQ helix (gray) represents the functional interaction between the C-terminal tail complex and the CaVβ2a–AID complex required for CDI and CDF. In CaV1.2 GGG (bottom), IS6-AID helix disruption blunts the influence of the Ca2+/CaM-IQ domain on the transmembrane pore.
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