Ankyrin-B Regulates Cav2.1 and Cav2.2 Channel Expression and Targeting*

Background: Calcium channels control membrane excitability. The mechanisms underlying Cav2.1/Cav2.2 targeting are not well understood. Results: Ankyrin-B associates with Cav2.1/Cav2.2. Loss of ankyrin-B results in reduced expression of Cav2.1/Cav2.2 in select brain regions. Conclusion: Ankyrin-B plays a role in the expression of Cav2.1/Cav2.2. Significance: Results identify pathway for membrane targeting of calcium channels and regulation of membrane excitability. 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.

Neurons are highly polarized cells, where structural, functional, and molecular differences within the neuronal physiology underlie the ability to receive, process, and transmit information. The specific expression profile of ion channels, receptors, and cytoskeletal proteins present within select neuronal membrane domains plays a primary role in defining local function of the entire cell. Recently, studies focused on the role of cytoskeletal proteins in the genesis and maintenance of local membrane domains in excitable cells have revealed a new and exciting avenue of study for these proteins in physiology and disease (1,2).
Calcium ions are critical second messengers involved in the regulation of multiple neuronal cellular processes, but primarily synaptic transmission (3)(4)(5)(6)(7). Ca 2ϩ entry across the plasma membrane occurs via voltage-dependent calcium channels (VDCCs). 2 To maintain the tight spatio-temporal distribution of intracellular Ca 2ϩ necessary to mediate a diverse array of functions, VDCCs have evolved with respect to their biophysical properties, regulation, and subcellular localization (8). The physiological importance of VDCCs is further exemplified by the variety of mutations in VDCC channel components linked to neurological and motor diseases (9). VDCCs have been classified into five groups, identified as T, L, N, P/Q, and R, based on electrophysiological and pharmacological properties (9 -12). N-and P/Q-type currents are observed primarily in neurons and co-localize with a subset of docked vesicles at the synapse, where they control exocytosis (13)(14)(15) and initiate synaptic neurotransmission (3,4,16). VDCCs are heteromultimers composed of the pore-forming ␣1 subunit and associated auxiliary subunits, Ca v ␤ and ␣2␦ (for review, see Ref. 4). Although auxiliary subunits have been demonstrated to enhance ␣1B expression (17,18), the mechanisms underlying Ca v channel membrane expression and localization are not clearly defined.
Ankyrins are a class of membrane adaptor proteins necessary for the targeting and regulation of select membrane and cytoplasmic proteins (19). In the brain, ankyrin-G is a necessary component for the functional organization of initial segments and nodes of Ranvier. Targeted disruption of cerebellar ankyrin-G results in severe ataxia, loss of ability to fire action potentials, and loss of Na v 1.6 from the initial segment (20,21). Additionally, there is loss of nodal ␤ IV spectrin and KCNQ2/3 channels with indiscriminate localization of the cell adhesion molecule neurofascin (21,22). Loss of neurofascin expression at the initial segment of Purkinje neurons results in disruption of interneuron synapses connecting Purkinje neurons in the cerebellum (23). Likewise, ankyrin-B has been demonstrated to play a critical role in the establishment and maintenance of excitable domains in brain (24). Loss of ankyrin-B in mice results in significant nervous system defects, including hypoplasia of the corpus callosum and pyramidal tracts, dilation of the lateral ventricles, and deterioration of long axon tracts (25). Notably, recent work outside of the brain has defined new roles for ankyrin-B for targeting select voltage-gated Ca 2ϩ channels in the pacemaking cells of the cardiac sinoatrial node (26,27). We therefore tested the role of ankyrin-B for targeting select calcium channels in the brain.
Our findings define a role for ankyrin-B in neuronal cell biology by demonstrating ankyrin-B-dependent interactions that are required for proper Ca v 2.1 and Ca v 2.2 expression in brain. We observed a significant decrease in Ca v 2.1 and Ca v 2.2 expression in cortex, cerebellum, and brain stem tissues of ankyrin-B-deficient mice. In defining the structural requirement for this interaction, we demonstrate that the ankyrin-B membrane-binding domain associates directly with Ca v 2.

EXPERIMENTAL PROCEDURES
Animals-Mice were age-matched wild-type, ankyrin-B ϩ/Ϫ , and ankyrin-G cerebellar-specific knock-out littermates. Animals were backcrossed at least 20 generations (99.8% pure) into the C57BL/6 background (Jackson Laboratories). Ankyrin-G cerebellar-specific knock-out mice were generated as described (25). Animals were handled according to approved protocols and animal welfare regulations of the Institutional Animal Care and Use Committee at Ohio State University. All mice were housed and handled identically.
Tissue Preparation and Homogenization-For immunoblot and co-immunoprecipitation (co-IP) analysis, brain tissues (cortex, cerebellum, and brain stem) were flash-frozen in liquid nitrogen and ground into a fine powder. The powder was resuspended in 2 volumes of ice-cold homogenization buffer (50 mM Tris-HCl (pH 7.35), 10 mM NaCl, 0.32 M sucrose, 5 mM EDTA, 2.5 mM EGTA, 1 mM PMSF, 1 mM AEBSF, 10 g/ml leupeptin, and 10 g/ml pepstatin) and homogenized using a hand-held homogenizer (27,28). The homogenate was centrifuged at 1000 ϫ g at 4°C to remove nuclei. Triton X-100 and deoxycholate were added to the postnuclear supernatant for final concentrations of 0.75% Triton X-100 and 1% deoxycholate.
The lysate was pelleted at high speed for 15 min at 4°C. The resulting supernatant was quantitated by bicinchoninic acid assay prior to analysis.
Immunoblots-Immunoblots from anti-ankyrin-B, anti-Ca v 2.1, anti-Ca v 2.2, and anti-ankyrin-G blots were evaluated by densitometry and expression normalized to anti-GAPDH blots (29,30). Histograms represent expression as a percentage of wildtype expression (wild-type expression normalized to 100%).
Tissue Preparation for Immunostaining-Freshly extracted tissues from wild-type and ankyrin-B ϩ/Ϫ mice were fixed in 4% paraformaldehyde for 12 h and the tissues transferred to 10, 20, and 30% sucrose solutions for 12 h each. Tissues were cryosectioned to 10-m thickness. Cryosections were rehydrated in PBS prior to preblocking in 3 mg/ml BSA in PBS. Primary antibodies were made in a vehicle of 3 mg/ml BSA with 0.1% Triton X-100 in PBS and incubated on sections overnight at 4°C. The slides were washed three times in vehicle and incubated with Alexa Fluor donkey anti-rabbit 568 secondary antibodies for 3 h at 4°C. Following three washes in vehicle, the slides were mounted with VectaShield (Vector Laboratories) and no. 1 coverslips. Images were collected on a Zeiss 510 Meta confocal microscopy using Carl Zeiss software.  FEBRUARY 21, 2014 • VOLUME 289 • NUMBER 8

JOURNAL OF BIOLOGICAL CHEMISTRY 5287
Co-IP from Brain Lysates-Protein A-conjugated agarose beads (AffiGel; Bio-Rad) were incubated with either control Ig or anti-Ca v 2.2 Ig, anti-Ca v 2.1 Ig, or anti-ankyrin-B Ig in co-IP binding buffer (PBS with 0.1% Triton X-100 and protease inhibitor mixture (Sigma)) for 12 h at 4°C. Beads were centrifuged and washed three times in ice-cold PBS. Wild-type cortex, cerebellum, or brain stem tissue lysate were added to the washed beads, along with protease inhibitor mixture and co-IP binding buffer, and incubated for 12 h at 4°C. The reactions were washed three times in ice-cold co-IP buffer. The samples were eluted and the proteins separated by SDS-PAGE prior to immunoblots with ankyrin-B, Ca v 2.1, or Ca v 2.2 Ig. Experiments were performed multiple times with similar results. Due to the low copy number of Ca v 2.1 and Ca v 2.2 in brain regions, lysate inputs were scaled up. For experiments where Ca v 2.1 or Ca v 2.2 Igs were immobilized on beads, 1 mg of cortex and cerebellum lysate was used, whereas 2 mg of brain stem lysate was used. Input lanes represent 10% of total lysate used. For experiments where ankyrin-B Ig was immobilized on beads, 1 mg of lysate for each brain region was utilized. Input lanes represent 5% of total lysate for cortex and cerebellum and 10% of total lysate for brain stem.
Co-IP from Transfected Cells-Protein A-conjugated agarose beads were incubated with either control IgG or affinity-purified anti-ankyrin-B Ig for 12 h at 4°C. Beads were centrifuged and washed three times in ice-cold PBS. 100 g of transfected HEK lysate was added to the washed beads, along with protease inhibitor mixture and IP buffer, and incubated for 12 h at 4°C. The reactions were washed three times in ice-cold IP buffer. The samples were eluted and the proteins separated by SDS-PAGE prior to immunoblots with anti-GFP Ig. For co-IP using full-length calcium channel-transfected cells, input lanes represent 20% of total lysate input (400 g). For GFP-Ca v channel loop transfections, input lanes represent 10% of total lysate input (200 g).
Pulldowns-One hundred micrograms of wild-type cortex, cerebellum, or brain stem lysate was added to 10 g of GST, GST-ankyrin-B MBD, GST-ankyrin-B SBD, or GST-ankyrin-B CTD-conjugated beads, along with 500 l of Binding Buffer, protease inhibitor mixture (Sigma), and PMSF. The reactions incubated for 12 h at 4°C. The beads were centrifuged and washed three times in binding buffer. The proteins were eluted, separated by SDS-PAGE, and immunoblotted using anti-Ca v 2.1 Ig and anti-Ca v 2.2 Ig.
HEK293 Cell Culture and Transfection of Ca v 2.1 and Ca v 2.2 Fragments-HEK293 cells were maintained in Dulbecco's modified essential medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; HyClone) and 0.1% penicillin/ streptomycin. Cells were cultured at 37°C in 5% CO 2 . Cultured cells were split 24 h prior to transfection and plated to obtain 30% confluence at time of transfection. Effectene reagent (Qiagen) was used to transfect cells with 0.2 g Ca v 2.2 DII/III pEGFP-C3, Ca v 2.2 DII/III Y788E pEGFP-C3, or pEGFP-C3 alone. Transfection was carried out for 7 h at 37°C and the cells recovered overnight. After 48 h, the cells were fixed with 2% paraformaldehyde, stained with ToPro (to discriminate nuclear material), and evaluated by confocal microscopy. Likewise, transfections using Ca v 2.1 DII/III pEGFP-C3, Ca v 2.1 DII/III Y797E pEGFP-C3, and pEGFP-C3 alone were also performed using the same protocol.
HEK293 Cell Culture and Transfection for Confocal Microscopy-HEK293 cells were maintained in Dulbecco's modified essential medium supplemented with 10% FBS and 0.1% penicillin/streptomycin. Cells were cultured at 37°C in 5% CO 2 . Cultured cells were split 24 h prior to transfection and plated on MatTek plates to obtain 90% confluence at time of transfection. Lipofectamine (Invitrogen) was used to transfect cells with 1 g of Ca v 2.2-GFP, 1 g of Ca v 2.2 Y788E-GFP, 1 g of Ca v 2.1-GFP, 1 g of Ca v 2.1 Y797E-GFP, or pAcGFP1-N2 alone. Transfection was carried out for 5 h at 37°C and the cells recovered overnight. After 24 h, the cells were fixed with 2% paraformal- dehyde, mounted with VectaShield (with DAPI), and evaluated by confocal microscopy.
HEK293 Cell Culture and Transfection for Co-immunoprecipitations-HEK293 cells were maintained in Dulbecco's modified essential medium supplemented with 10% FBS and 0.1% penicillin/streptomycin. Cells were cultured at 37°C in 5% CO 2 . Cultured cells were split 24 h prior to transfection and plated on 100-mm 2 plates to obtain 90% confluence at time of transfection. Lipofectamine (Invitrogen) was used to transfect cells with 1 g of Ca v 2.2-GFP, 1 g of Ca v 2.2 Y788E-GFP, 1 g of Ca v 2.1-GFP, 1 g of Ca v 2.1 Y797E-GFP, or pAcGFP1-N2 alone. Untransfected cells were used as a negative control. Transfection was carried out for 5 h at 37°C and the cells recovered overnight. After 24 h, the cells were lysed in IP buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 5% glycerol, protease inhibitor mixture (Sigma)) and centrifuged to remove cellular debris, and the protein was quantitated by bicinchoninic acid assay (Pierce).
Statistics-Data are presented as a mean Ϯ S.D., and statistical significance was assessed using Student's t test. The null hypothesis was rejected for p Ͻ 0.05.
Based on our immunoblot findings, we performed confocal analysis for the localization of Ca v 2.1 and Ca v 2.2 in wild-type and ankyrin-B ϩ/Ϫ mouse cerebellum. In the cerebellum, Ca v 2.1 and Ca v 2.2 are expressed in Purkinje neurons and localized in the cell body (31,32). Ankyrin-B is also expressed in the cerebellar Purkinje neuron cell body, as well as throughout the molecular layer (33). As shown in Fig. 2, whereas ankyrin-B, Ca v 2.1, and Ca v 2.2 are robustly expressed in Purkinje neurons from wild-type cerebellum, we observed a significant decrease  A and B, n ϭ 4; N.S.). Ankyrin-G levels by immunoblotting in ankyrin-G cerebellar-specific knock-out mice were significantly reduced and similar to background.   (Fig. 2).

Ankyrin-B Regulates Ca v Channel Targeting
Ankyrin-G Is Not Required for Ca v 2.1 or Ca v 2.2 Expression-Although encoded from different genes, ankyrin-B and ankyrin-G display similar domain organization, and both target ion channels and transporters in diverse excitable cell types (2). As both ankyrin-B and ankyrin-G are co-expressed in cell types harboring Ca v 2.1 and Ca v 2.2, we evaluated whether ankyrin-G is required for Ca v 2.1 and Ca v 2.2 expression as shown for ankyrin-B (Figs. 1 and 2). In contrast to findings for ankyrin-Bdeficient tissue, we observed no difference in Ca v 2.1 or Ca v 2.2 expression in cerebellar lysates generated from a well characterized mouse model homozygous for a null mutation in cerebellar ankyrin-G ( Fig. 3 and Ref. 20). These data demonstrate that ankyrin-B, but not ankyrin-G, is required for regulating Ca v 2.1 and Ca v 2.2 expression in the cerebellum. Moreover, these data suggest that ankyrins-G and -B play nonredundant roles in calcium channel targeting in brain.
Ankyrin-B Associates with Ca v 2.1 and Ca v 2.2 in Brain-Considering that Ca v 2.1 and Ca v 2.2 expression levels were significantly reduced in ankyrin-B ϩ/Ϫ brain tissue (Figs. 1 and 2), we evaluated the ability of ankyrin-B to associate with Ca v 2.1 and Ca v 2.2 in brain. Co-IP analysis using detergent-soluble lysates from adult mouse cortex, cerebellum, and brain stem demonstrated that affinity-purified anti-Ca v 2.1 Ig co-immunoprecipitated ankyrin-B from cortex (Fig. 4A), cerebellum (Fig. 4B), and brain stem (Fig. 4C). Likewise, anti-Ca v 2.2 Ig co-immunopre-cipitated ankyrin-B from cortex, cerebellum, and brain stem lysates (Fig. 4, D-F). We observed no interaction between ankyrin-B and control Ig in any tissue lysate (Fig. 4, A-F). To confirm this interaction, we performed reciprocal co-immunoprecipitations utilizing affinity-purified ankyrin-B Ig. Ankyrin-B Ig co-immunoprecipitated Ca v 2.1 and Ca v 2.2 from detergent-soluble lysates from cortex, cerebellum, and brain stem, whereas we observed no interaction with a control Ig (Fig. 4, G-L). Collectively, these data support an in vivo interaction between ankyrin-B and Ca v 2.1, as well as an in vivo interaction between ankyrin-B and Ca v 2.2, in mouse cortex, cerebellum, and brain stem.
We further evaluated the neuronal ankyrin-B/Ca v 2.1 and ankyrin-B/Ca v 2.2 interactions using pulldown experiments from mouse detergent-soluble cortex, cerebellum, and brain stem lysates. Ankyrin-B comprises three distinct structural domains: MBD, SBD, and CTD (Fig. 5A). Of note, GSTankyrin-B MBD, but neither GST-ankyrin-B SBD or GSTankyrin-B CTD interacted with Ca v 2.1 nor Ca v 2.2 from cortex (Fig. 5B), cerebellum (Fig. 5C), and brain stem ( Fig. 5D; fusion proteins are shown in Fig. 5E). Collectively, these data demonstrate an interaction between Ca v 2.1 and Ca v 2.2 and ankyrin-B within multiple regions of the brain. These data further identify the structural requirements on ankyrin-B for Ca v channel binding as the ANK repeats of the membrane-binding domain.
Ankyrin  suggested that a region in the DII/III loop region of Ca v 2.2 contains a region postulated to play a role in the targeting of calcium channels (34) (Fig. 6, A and B). This DII/III region contains a 20-amino acid motif that is highly conserved among species (Fig. 6, C and D). Three constructs based on Ca v 2.1 and Ca v 2.2 sequences were designed to evaluate the role of this motif in ankyrin-B/Ca v 2.1 and ankyrin-B/Ca v 2.2 association (Fig. 6, E and F). The first construct encompassed the entire DII/III loop (Ca v 2.1 amino acids 708 -1194; Ca v 2.2 amino acids 710 -1139); the second initiated proximal to the putative ankyrin-binding motif (ABM; Ca v 2.1 amino acids 788 -1194; Ca v 2.2 amino acids 779 -1139); and the third began distal to the ABM (Ca v 2.1 amino acids 808 -1194; Ca v 2.2 amino acids 799 -1139). In vitro binding with immobilized GST-ankyrin-B MBD revealed that the full-length Ca v 2.1 DII/III loop and the Ca v 2.1 construct containing residues 788 -1194 displayed binding activity for GST-ankyrin-B MBD (Fig. 6G). In contrast, the Ca v 2.1 construct lacking residues 788 -808 (construct 808 -1194) had binding activity similar to GST alone (Fig. 6G). Similarly, full-length Ca v 2.2 DII/III loop and the Ca v 2.2 construct containing residues 779 -799 (construct 779 -1139) displayed binding activity for GST-ankyrin-B MBD (Fig. 6H). The Ca v 2.2 construct lacking residues 779 -799 (construct 799 -1139) had binding activity similar to GST alone (Fig. 6H). These data identify a 20-amino acid motif in both the Ca v 2.1 and Ca v 2.2 DII/III loops that is necessary for interaction with ankyrin-B.
A Conserved Tyrosine Residue Regulates Association with Ankyrin-B-Although inhibition of calcium channels is voltage-dependent, it is mediated by direct interaction of G protein ␤␥ subunits with the ␣1 pore-forming subunit of the channel (35,36). Additionally, phosphorylation by tyrosine kinases has been shown to inhibit calcium channels (37) with G protein-dependent inhibition of calcium current due in part to a decrease in the open probability of the channel, resulting in reduced current density (38 -40). We had previously defined a 20-amino acid motif in the DII/III loops of Ca v 2.1 and Ca v 2.2 that associates with ankyrin-B (Fig. 6). Notably, this motif contains a conserved tyrosine residue. To evaluate the role of this tyrosine in the regulation of Ca v 2.1 and Ca v 2.2 association with ankyrin-B, we incorporated a Y797E mutation in the Ca v 2.1 DII/III loop and evaluated the effect on in vitro binding (Fig. 7A). Mutagenesis of the Tyr-797 residue to a glutamic acid resulted in loss of association with GST- ankyrin-B MBD by in vitro binding analysis (Fig. 7B). Mutation of the analogous residue, Tyr-788, in the Ca v 2.2 DII/III to a glutamic acid (Fig. 7D) demonstrated a similar loss of in vitro binding with GST-ankyrin-B MBD (Fig. 7E).

DII/DIII Tyrosine Residues Are Critical for Ca v 2.1/Ca v 2.2
Targeting-Ankyrin proteins are critical for targeting ion channels and transporters in diverse cell types including neurons. In fact, ion channel or transporter ankyrin-binding motifs alone  are sufficient to target GFP and other markers in heterologous cells and primary cultures (41). We therefore tested (i) whether the DII/DIII loops of Ca v 2.1 and Ca v 2.2 (that harbor ankyrinbinding motifs) were sufficient to behave as a dominant target signal in HEK293 cells, and (ii) whether the single tyrosine mutation in each channel altered this trafficking pattern. DII/ DIII loops from Ca v 2.1 and Ca v 2.2 were subcloned in-frame into the pEGFP-C3 expression vector and transfected into HEK293 cells (express endogenous ankyrin-B). Confocal analysis revealed that unlike GFP that was exclusively found in the HEK293 cell nucleus (Fig. 8, A and D), GFP-Ca v 2.1 DII/III loop and GFP-Ca v 2.2 DII/III were in fact targeted outside of the nucleus (versus GFP) and had a diffuse pattern of expression throughout the cell (Fig. 8, B and E). Consistent with these findings, both GFP-Ca v 2.1 DII/III loop and GFP-Ca v 2.2 DII/III loop interacted with ankyrin-B in parallel co-IP experiments from transfected cells (Fig. 8, G-I). In contrast, GFP-Ca v 2.1 DII/III Y797E and GFP-Ca v 2.2 DII/III Y788E lacked ankyrin-Bbinding activity (Fig. 8, G-I) and were restricted in localization to a tight and consistent perinuclear distribution (Fig. 8, C  We confirmed these findings in the context of full-length Ca v 2.1 and Ca v 2.2. We observed both GFP-Ca v 2.1 and GFP-Ca v 2.2 with diffuse expression across transfected HEK293 cells, whereas GFP-Ca v 2.1 Y797E and full-length GFP-Ca v 2.2 Y788E displayed abnormal targeting with a clustered perinuclear distribution (Fig. 9, A-E). Consistent with these findings, whereas GFP-Ca v 2.1 and GFP-Ca v 2.2 associated with ankyrin-B in co-IP experiments, ankyrin-B Ig was unable to co-immunoprecipitate either full-length GFP-Ca v 2.1 Y797E or full-length GFP-Ca v 2.2 Y788E from HEK293 cell lysates (Fig. 9, F-H). These results further support a critical role for ankyrin-B-mediated Ca v 2.1 and Ca v 2.2 expression and targeting.

DISCUSSION
Our findings demonstrate a role of ankyrin-B for Ca v 2.1 and Ca v 2.2 channel expression and targeting in brain. Loss of ankyrin-B results in decreased expression of Ca v 2.1 and Ca v 2.2 in cortex, cerebellum, and brain stem. Moreover, mutation of a single residue in the Ca v 2.1 and Ca v 2.2 DII/III loop results in loss of association with ankyrin-B and abnormal cellular targeting. Ankyrin-B has been demonstrated to play a critical role in the establishment and maintenance of excitable domains in brain. Loss of ankyrin-B in mice results in significant nervous system defects, including hypoplasia of the corpus callosum and pyramidal tracts, dilation of the lateral ventricles, and deterioration of long axon tracts (25). Specifically, these phenotypes are associated with loss of L1CAM proteins throughout the brain. Interestingly, mutations in the human L1CAM gene cause mental retardation as well as a collection of neurological symptoms referred to as CRASH syndrome (corpus callosum hypoplasia, retardation, aphasia, spastic paraplegia, and hydrocephalus) (42). The ankyrin-B Ϫ/Ϫ mouse model, although lethal at post-natal day 1, exhibits loss of L1CAM from axons and exhibits many of the CRASH symptoms, although with greater severity (25). No humans with null mutations in the ankyrin-B gene (ANK2) have been identified; however, given that heterozygote individuals with loss-of-function mutations in the ANK2 gene are present in the population (43) and are found in patients with cardiac arrhythmia phenotypes (26, 44 -47), it is possible that rare homozygous or mixed heterozygotes may exhibit CRASH symptoms. S1224L and Y1229H mutations in the human L1CAM gene are associated with CRASH syndrome (48). Moreover, these mutations are located within the highly conserved ankyrin-binding site and abolish association of L1 with ankyrin-B (48).
Ca v 2.1 and Ca v 2.2 channel activity in brain is necessary for proper initiation of synaptic transmission. Ca v 2.2 Ϫ/Ϫ animals demonstrate normal motor coordination; however, these mice exhibit reduced responses to pain stimuli (49) and elevated aggression (50). Ca v 2.1 Ϫ/Ϫ animals demonstrate dystonia at postnatal day 10 -12 and rarely survive past weaning (51). Likewise, Ca v 2.1 Ϫ/Ϫ mice exhibit reduced responses to pain stimuli (52). As noted above, ankyrin-B Ϫ/Ϫ die at birth, therefore we currently lack data on the in vivo phenotypes of ankyrin-B deficiency in an adult mouse. Future studies utilizing inducible ankyrin-B knock-out strategies in the postnatal mouse will be important for understanding the role of this cytoskeletal protein in synaptic function.
Finally, although our data link ankyrin-B with regulation of targeting via the DII/DIII loop of both Ca v 2.1 and Ca v 2.2 via specific tyrosine residues, it is possible that other protein interactions in this loop may also play critical roles in ion channel processing and membrane regulation. For example, the Ca v 2.2 DII/DIII loop maintains a SNARE binding or synprint region that has been linked with Ca v channel targeting (34). It will be important in future work to understand the potential dual regulation of Ca v channel targeting by ankyrin and other proteins.