Ankyrin-B regulates Cav2.1 and Cav2.2 channel expression and targeting

Abstract

N-type and P/Q-type calcium channels are documented players in the regulation of synaptic function; however, the mechanisms underlying their expression and cellular targeting are poorly understood. Ankyrin polypeptides are essential for normal integral membrane protein expression in a number of cell types, including neurons, cardiomyocytes, epithelia, secretory cells, and erythrocytes. Ankyrin dysfunction has been linked to defects in integral protein expression, abnormal cellular function, and disease. Here, we demonstrate that ankyrin-B associates with Cav2.1 and Cav2.2 in cortex, cerebellum, and brain stem. Additionally, using in vitro and in vivo techniques, we demonstrate that ankyrin-B, via its membrane-binding domain, associates with a highly conserved motif in the DII/III loop domain of Cav2.1 and Cav2.2. Further, we demonstrate that this domain is necessary for proper targeting of Cav2.1 and Cav2.2 in a heterologous system. Finally, we demonstrate that mutation of a single conserved tyrosine residue in the ankyrin-binding motif of both Cav2.1 (Y797E) and Cav2.2 (Y788E) results in loss of association with ankyrin-B in vitro and in vivo. Collectively, our findings identify an interaction between ankyrin-B and both Cav2.1 and Cav2.2 at the amino acid level that is necessary for proper Cav2.1 and Cav2.2 targeting in vivo.

Keywords: Adaptor Proteins; Cytoskeleton; Ion Channels; Membrane Trafficking; Protein Sorting; Protein Targeting.

FIGURE 1.

Ca_v2.1 and Ca_v2.2 expression levels are decreased in ankyrin-B^+/− brain lysates. Ankyrin-B, Ca_v2.1, and Ca_v2.2 expression levels are significantly decreased in cortex, cerebellum, and brain stem in ankyrin-B^+/− mice compared with wild-type mice (A, C, and D, respectively). There are no significant differences in expression between wild-type and ankyrin-B^+/− lysates of ankyrin-G (B) or GAPDH (E, loading control). For each panel, n = 4 for each genotype (p < 0.05).

FIGURE 2.

Ankyrin-B^+/− mice display a significant decrease in ankyrin-B, Ca_v2. 1, and Ca_v2.2 expression in cerebellar Purkinje neurons. A and B, ankyrin-B expression is significantly reduced in ankyrin-B^+/− Purkinje neurons. Note that ankyrin-B is expressed in both Purkinje cell layer and molecular layer. C–F, Ca_v2.1 (C and D) and Ca_v2.2 (E and F) expression is significantly reduced in the Purkinje neurons of ankyrin-B^+/− mice. Scale bar denotes 20 μm. G and H, Ca_v2.1 and Ca_v2.2 antibodies are validated. Untransfected HEK293 cells and HEK293 cells transfected with either full-length GFP-Ca_v2.1 or GFP-Ca_v2.2 were analyzed by co-immunoprecipitation or immunoblotting (IB). In G, Ca_v2.1 Ig recognizes GFP-Ca_v2.1 but not GFP-Ca_v2.2. In H, Ca_v2.2 Ig recognizes GFP-Ca_v2.2 but not GFP-Ca_v2.1.

FIGURE 3.

Ankyrin-G is not required for Ca_v2.1 or Ca_v2.2 expression. Although ankyrin-G cerebellar-specific knock-out mice display a significant reduction in the expression of ankyrin-G (A), there are no significant differences in the expression of Ca_v2.1 or Ca_v2.2 between wild-type and ankyrin-G cerebellar-specific knock-out cerebellar lysates (A and B, n = 4; N.S.). Ankyrin-G levels by immunoblotting in ankyrin-G cerebellar-specific knock-out mice were significantly reduced and similar to background.

FIGURE 4.

Ankyrin-B co-immunoprecipitates with Ca_v2.1 and Ca_v2.2. A–F, Ca_v2.1 Ig and Ca_v2.2 Ig co-immunoprecipitate ankyrin-B from detergent-soluble lysates of cortex (A and D), cerebellum (B and E), and brain stem (C and F), respectively. No significant interaction was observed with control (Ctrl) Ig. G–L, ankyrin-G Ig co-immunoprecipitates Ca_v2.1 and Ca_v2.2 from detergent-soluble lysates of cortex (G and J), cerebellum (H and K), and brain stem (I and L), respectively. No significant interaction was observed with control Ig. IB, immunoblotting.

FIGURE 5.

Ankyrin-B associates with Ca_v2.1 and Ca_v2.2. A, ankyrin-B comprises a membrane-binding domain, spectrin-binding domain, and a C-terminal regulatory domain. B–D, GST-ankyrin-B MBD associates with both Ca_v2.1 and Ca_v2.2 from detergent-soluble lysates of cortex (B), cerebellum (C), and brain stem (D). No interaction was observed between lysates and GST-ankyrin-B SBD, GST-ankyrin-B CTD, or GST alone. E, GST fusion proteins were utilized for pull-down experiments. Fusion proteins were purified from bacteria and analyzed by Coomassie Blue staining. Whereas GST migrates at 29 kDa, GST-ankyrin-B MBD migrates at 94 kDa. GST-ankyrin-B SBD migrates at 62 kDa, and GST-ankyrin-B CTD migrates at 79 kDa.

FIGURE 6.

Ca_v2.1 and Ca_v2.2 DII/DIII motif associates with ankyrin-B. A and B, location of putative ABM in the DII/DIII cytoplasmic loop of Ca_v2.1 and Ca_v2.2 is shown. C and D, the putative ABM in Ca_v2.1 and Ca_v2.2 is conserved across species and harbors a central conserved tyrosine (bolded). E and F, three constructs were designed to evaluate the role of this amino acid sequence in ankyrin-B binding. These constructs include the entire DII/III loop, a portion of the loop that begins just prior to the putative ABM, and a portion of the loop that begins just after the putative ABM. G and H, in vitro binding assays demonstrate that, whereas the full-length DII/III loops of Ca_v2.1 and Ca_v2.2 and the smaller loop portion that contains the putative ABM are able to associate with GST-ankyrin-B MBD, the smaller loop portion that lacks this domain demonstrates no binding with GST-ankyrin-B MBD. I and J, Coomassie Blue staining demonstrating that equal loading of GST constructs was performed prior to imaging (denoted by asterisks).

FIGURE 7.

A conserved tyrosine residue in the Ca_v2. 1 and Ca_v2.2 DII/III loop is necessary for ankyrin-B interaction in vitro. A and D, Ca_v2.1 and Ca_v2.2 DII/III loop domains contain a conserved tyrosine residue that was experimentally mutated to a glutamic acid to assess ankyrin-B binding. B, GST-ankyrin-B MBD was able to associate with the Ca_v2.1 DII/III loop domain. In contrast, there was a loss of interaction with the domain containing the Y797E mutation. There was no appreciable binding with GST alone. E, Ca_v2.2 DII/III loop domain associated with GST-ankyrin-B MBD is shown. However, there was no appreciable association between GST-ankyrin-B MBD and the Ca_v2.2 DII/III Y788E. There was no detectable binding to GST alone. C and F, Coomassie Blue stains demonstrate that equal loading of GST constructs was performed prior to imaging.

FIGURE 8.

Ca_v2.1/Ca_v2.2 tyrosine residues in ABM are critical for cellular targeting. HEK293 cells transfected with pEGFP-C3 demonstrated a primarily nuclear localization pattern (A and D), whereas HEK293 cells transfected with Ca_v2.1 DII/III-GFP (B) or Ca_v2.2 DII/III-GFP (E) displayed a diffuse cellular pattern of expression unique from the nucleus. C and F, unlike WT constructs, both Ca_v2.1 Y797E-GFP and Ca_v2.2 Y788E-GFP were distributed in a tight perinuclear pattern. Scale bars denote 8 μm. G–I, a conserved tyrosine residue in the Ca_v2.1 and Ca_v2.2 DII/III loop is necessary for ankyrin-B interaction in vitro. G, in control experiments, ankyrin-B Ig did not interact with GFP alone expressed in HEK293 cells. H and I, ankyrin-B Ig co-immunoprecipitates GFP fusion protein containing the Ca_v2.1 or Ca_v2.2 DII/DII loop from transfected HEK293 cells. In contrast, ankyrin-B Ig does not co-immunoprecipitate GFP fusion proteins of Ca_v2.1 DII/DIII Y797E or Ca_v2.2 DII/III Y788E in parallel experiments. IB, immunoblotting.

FIGURE 9.

Ca_v2. 1/Ca_v2.2 tyrosine residues in ABM are critical for cellular targeting and ankyrin interaction. A, HEK293 cells transfected with pEGFP-C3 demonstrated a primarily nuclear localization pattern. B and D, in contrast, HEK293 cells transfected with full-length GFP-Ca_v2.1 (B) or GFP-Ca_v2.2 (D) displayed a diffuse cellular pattern of expression unique from the nucleus or perinuclear region. C and E, unlike WT Ca_v2.1 and Ca_v2.2 constructs, both full-length GFP-Ca_v2.1 Y797E and full-length GFP-Ca_v2.2 Y788E-GFP were distributed in a tight perinuclear pattern (white arrowheads). Scale bars denote 8 μm. F, control experiments demonstrate that ankyrin-B Ig does not associate with GFP alone. G, ankyrin-B co-immunoprecipitates full-length GFP-Ca_v2.1, but not full-length GFP-Ca_v2.1 Y797E from transfected HEK293 lysates. H, ankyrin-B co-immunoprecipitates full-length GFP-Ca_v2.2, but not full-length GFP-Ca_v2.2 Y788E from transfected HEK293 lysates.