2.2 Results
2.2.4 Molecular determinants for CaV1.3 NTD interaction with CaMKII
Previous studies indicate that the CaV1.2 and CaV1.3 NTDs contain conserved binding sites for calmodulin (residues S52-K64 in CaV1.3), termed NSCaTE (Dick et al., 2008;
Tadross et al., 2008), and for CaMKII (K110-W123 in CaV1.2) (Simms et al., 2014). To investigate the potential roles of these domains in the CaMKII binding detected here, we mapped the site of direct CaMKII interaction in the CaV1.3 NTD (Fig. 2.4A). There was no detectable interaction between pre-activated CaMKII and the membrane distal fragment (NT-A: amino acids 1-68) containing the NSCaTE domain (Fig. 2.4B), but the membrane proximal fragment (NT-B: amino acids 69-126) robustly interacts with pre- activated CaMKII. Further dissection of NT-B revealed that pre-activated CaMKII interacts with a GST-tagged fragment containing residues 69-93 (NT-B1), but not with two fragments containing more membrane proximal residues 94-110 or 111-126 (NT-B2 and NT-B3, Fig. 2.4C).
The amino acid sequence of CaV1.3 residues 69-93 shares little identifiable similarity with known CaMKII-binding domains in other proteins (Bayer et al., 2001; Grueter et al., 2008; Strack et al., 2000a). However, we identified three basic amino acids in CaV1.3 (Arg83-Lys84-Arg85) that are largely conserved in NTDs of CaV1.2 and other LTCC 1 subunits, but not in the NTDs of CaV2.2 or CaV3.2 (which do not bind CaMKII), or of other 1 subunits (Fig. 2.3A). Replacement of this RKR motif with three alanines in the CaV1.3 NTD almost completely abrogated binding of pre-activated CaMKII (Fig. 2.4D).
These data identify three amino acids in the CaV1.3 NTD that are required for strong and direct in vitro interactions with pre-activated CaMKII.
Comparison of the NTD sequences of CaV1 and CaV2 channels revealed that only one residue in the CaV1.3 RKR motif was not conserved in CaV2 channels (Fig. 2.3A). To test the importance of the each of the amino acids in CaMKII interaction, we created single and double alanine mutation of the R83KR85 and performed GST pulldown assay.
As shown in Fig. 2.4E and F, mutation of any one of the three amino acids is sufficient to disrupt the interaction between CaMKII and CaV1.3 N-terminal domain. Substitution of Lys84 or Arg85 with an alanine has a consistently stronger effect than substitution of Arg83, suggesting Lys84 and Arg85 are more critical in mediating CaMKII/CaV1.3 NTD interaction.
FIGURE 2.4 Characterization of the CaV1.3 NTD CaMKII binding domain. A.
Truncations used to map the CaMKII interaction site in the CaV1.3 NTD. Purple and white rectangles indicate approximate positions of previously-defined NSCaTE calmodulin-binding and CaMKII-binding domains, respectively (see legend to Fig. 1 and main text). B. Glutathione-agarose co-sedimentation assay comparing binding of activated CaMKII to the full-length CaV1.3 NTD, the membrane-distal part (NT-A) and the membrane-proximal part (NT-B). C. Analysis of further NTD truncations reveals that the NT-B1 region (residues 68-93) is sufficient for binding of activated CaMKII. D.
Mutation of amino acids R83KR85 to AAA within the full-length CaV1.3 NTD blocks CaV1.3-CaMKII interaction. E. and F. Further analysis and quantification of the effects of single or double mutation of the three basic amino acids R83KR85 on CaMKII binding.
These immunoblots are representative of at least three independent replicates.
Experiments in panels E and F were performed by Brynna Paulukaitis.
Preferential interactions of pre-activated CaMKII with several other CaMKII-associated proteins (CaMKAPs) are mediated by the catalytic domain. Therefore, to identify CaMKII residues critical for binding to the CaV1.3 NTD, we screened previously characterized as well as novel CaMKII mutations in the catalytic domain (Fig. 2.5A) using a fluorescence-based 96-well plate binding assay (see Methods). An I205K mutation, previously shown to disrupt binding to GluN2B and the densin-IN domain (Bayer et al., 2001; Bayer et al., 2006; Jiao et al., 2011), also reduced binding to the CaV1.3 NTD by ~80% (Fig. 2.5B). We identified two additional CaMKII mutations (V102E and E109K) that also significantly interfere with binding to the CaV1.3 NTD (Fig.
2.5B), whereas another mutation (Y210E) had no significant impact. Strikingly, the CaMKII-V102E mutation had no significant effect on binding to the 2a subunit of VGCCs, the densin-IN or -CTD domains (Fig 2.5C) or to GluN2B (not shown). In combination, these data suggest that the mechanism underlying binding of activated conformations of CaMKII to the CaV1.3 NTD is partially distinct from the mechanisms for binding to other known CaMKAPs.
FIGURE 2.5 Identification of a CaMKII mutation that specifically disrupts binding to the CaV1.3 NTD. A. CaMKII structures. Left, a single CaMKII subunit in an inactive (autoinhibited) conformation with an inhibitor (Bosutinib, yellow) bound in the nucleotide binding site (PDB:3SOA, (Chao et al., 2011)). Right, a single CaMKII subunit in an activated conformation (displaced regulatory domain) with a bound inhibitor (SU6656, yellow) (PDB:2WEL (Rellos et al., 2010)). The catalytic and regulatory domains are
shown in grey and pink, respectively. For clarity of presentation, C-terminal holoenzyme association domains are not shown and the displaced regulatory domain with bound Ca2+/calmodulin is not shown in PDB:2WEL. T286 and T305 (green) are two regulatory autophosphorylation sites. Mutation of I205/6 (orange) to Lys disrupts CaMKII interaction with GluN2B and Densin-IN (Bayer et al., 2001; Jiao et al., 2011), whereas mutation of D238/9 (cyan) to Arg disrupts GluN2B binding but spares interactions with Densin-IN (Jiao et al., 2011). A naturally occurring de novo E183 (purple) to Val mutation in CaMKII is linked to autism spectrum disorder and disrupts CaMKII interaction with multiple CaMKAPs (Iossifov et al., 2014; Stephenson et al., 2017). B. A 96-well glutathione plate assay to screen activated mApple-tagged CaMKII mutants for interactions with GST-tagged CaV1.3 NTD. C. Binding of activated mApple-tagged WT and V102E-CaMKII to multiple GST-CaMKAP proteins in the 96-well plate assay. A V102E mutation selectively disrupts CaMKII binding to the CaV1.3 NTD: V102/3 is highlighted in red in Panel A. Data from three independent experiments were analyzed by one-way ANOVA (for panel B) and two-way ANOVA followed by Sidak’s multiple comparison test (for panel C), respectively. ***, p<0.001; ns, not significant (p>0.05).
Experiments in panels B and C were performed by Christian Marks and Tyler Perfitt.
2.2.5 The CaV1.3 NTD is important for CaMKII association with LTCC complexes