AKAP79 modulation of L-type channels involves disruption of intramolecular interactions in the CaV1.2 subunit

Abstract

L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through β adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca(V)1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.(1) Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Ca(v)1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca(V)1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca(V)1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Ca(v)1.2 intracellular regions.

Figure 1. Substitution of Pro 857 by Ala in the Proline Rich Domain of the II-III loop and deletion of the two PRDs of the Ca_v2.1 C-terminus does not alter the AKAP79 modulation. (A) Conservation of the II-III-loop-PRD among the L type calcium channel family with the exception of Ca_V1.4 channels. (B) Schematic representation of the locations of PRDs along the Cav1.2 channel sequence, and amino acids sequence of the PRDs in the II-III linker and the C-terminus of Ca_V1.2. The top sequence is located in the domain II-III linker and starts at position 854. In the P875A mutant, Pro857 is replaced by Ala. The CΔPP mutant lacks the PEMPVGPRPRP stretch in the II-III linker. In the CΔP1P2 construct, the indicated proline rich regions at residues 1948 and 1967 are deleted. (C) Current amplitude ratio in the presence and absence of AKAP79 recorded for the different mutants. Current amplitude was taken at the peak of the I/V curve in each case and plotted as ratio between AKAP-injected and non-injected batches. Note that with the exception of the CΔPP mutant (without the poly-proline domain into the II-III loop), none of the other mutants are insensitive to AKAP79. Numbers in parenthesis indicate numbers of experiments.

Figure 2. Immunoprecipitation of Ca_V1.2 and AKAP79. (A) western blots of co-immunoprecipitations of Ca_V1.2-HA, calcium channel β₃-flag, and AKAP79-myc in 1% CHAPS using FLAG sepharose beads. The anti-flag antibody identifies the β₃-flag protein, while the AKAP79-myc protein is detected in the calcium channel complex indicating that AKAP79-myc binds to the calcium channel complex. (B) Ca_V1.2-HA is also immunoprecipitated by β₃-flag protein as expected.

Figure 3. AKAP79 directly binds to the leucine zipper motif in the C-terminus of the L-type calcium channel Ca_V1.2. (A) Amino acid sequence of the leucine zipper like motif in the C-terminal domain of Ca_V1.2. (B) Top: Schematic representation of the location of the II-III loop and the leucine zipper motif along the Ca_V1.2 channel sequence. Bottom: Yeast-two hybrid assay between AKAP79 and either the domain II-III linker, or the C-terminal leucine zipper motif of Ca_V1.2. pGAD was used as a negative control. Note the yeast growth in the leucine zipper/AKAP79 transformation. (C) In vitro binding of 6xHis Ca_V1.2 C-terminal and 6xHis Cav1.2 leucine zipper to immobilized AKAP79(GST). The western blot was probed using an anti-Xpress antibody (Invitrogen). Both the carboxy terminus and the Leucine Zipper motif bind to AKAP79.

Figure 4. Interaction between the Ca_V1.2 C-terminal and II-III linker region. (A) In vitro binding of 6xHis II-III loop of Ca_V1.2 to immobilized Ca_V1.2 GST C-terminus. The western blot was probed using the anti-Xpress antibody. Grouping of images from different parts of the same gel have been made. (B) Binding between 6xHis II-III loop of Ca_V1.2 and Ca_V1.2 GST-C-terminal in the presence of increasing AKAP79 levels. Note that AKAP79 prevents the II-III linker C-terminus association. Bottom: Control blot to show that identical amounts of GST C-terminal fusion protein were used in the assay.

Figure 5. AKAP mediated effect is isoform dependent. (A) Mean AKAP79 effect on current amplitude recorded for Ca_V1.2 wt and a leucine zipper deletion mutant Ca_V1.2-LZ. Note that AKAP79 does not increase current amplitudes of Ca_V1.2 channels lacking the leucine zipper. Right panel: western blot of in vitro binding assay between AKAP79 and the C-terminal domain of Ca_V1.2 lacking the leucine zipper motif. Note that the Ca_V1.2-LZ C-terminus region cannot bind AKAP79 in vitro. (B) Effect of AKAP18 on the Ca_V1.2 current amplitude. AKAP18 does not mediate an increase in current amplitude, but acts as a dominant negative to suppress the AKAP79 mediated current enhancement.