Wendy S. Pratt

The Calcium Channel α2δ-2 Subunit Partitions with CaV2.1 into Lipid Rafts in Cerebellum: Implications for Localization and Function

The accessory α2δ subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel α1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar α2δ-2 subunits with α2δ-2 stably expressed in tsA-201 cells. We identified that α2δ-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel α1 subunit CaV2.1, although CaV2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, α2δ-2 is not completely proteolytically processed into α2-2 and δ-2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which α2δ-2 also colocalizes with CaV2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced CaV2.1/α2δ-2/β4 currents. Furthermore, α2δ-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of α2δ-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable α2δ-2 cell line. In contrast, α2δ-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant α2δ-2 shows reduced functionality in terms of enhancement of CaV2.1/β4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of α2δ-2. Together, these results indicate that both α2δ-2 and CaV2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality.

The α2δ subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational modification essential for function

Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the α1 subunit is associated with two auxiliary subunits, the intracellular β subunit and the α2δ subunit; both of these subunits influence the trafficking and properties of CaV1 and CaV2 channels. The α2δ subunits have been described as type I transmembrane proteins, because they have an N-terminal signal peptide and a C-terminal hydrophobic and potentially transmembrane region. However, because they have very short C-terminal cytoplasmic domains, we hypothesized that the α2δ proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to δ rather than a transmembrane domain. Here, we provide biochemical, immunocytochemical, and mutational evidence to show that all of the α2δ subunits studied, α2δ-1, α2δ-2, and α2δ-3, show all of the properties expected of GPI-anchored proteins, both when heterologously expressed and in native tissues. They are substrates for prokaryotic phosphatidylinositol-phospholipase C (PI-PLC) and trypanosomal GPI-PLC, which release the α2δ proteins from membranes and intact cells and expose a cross-reacting determinant epitope. PI-PLC does not affect control transmembrane or membrane-associated proteins. Furthermore, mutation of the predicted GPI-anchor sites markedly reduced plasma membrane and detergent-resistant membrane localization of α2δ subunits. We also show that GPI anchoring of α2δ subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when α2δ subunits are coexpressed. In conclusion, this study redefines our understanding of α2δ subunits, both in terms of their role in calcium-channel function and other roles in synaptogenesis.

Dominant-Negative Calcium Channel Suppression by Truncated Constructs Involves a Kinase Implicated in the Unfolded Protein Response

Expression of the calcium channel CaV2.2 is markedly suppressed by coexpression with truncated constructs of CaV2.2. Furthermore, a two-domain construct of CaV2.1 mimicking an episodic ataxia-2 mutation strongly inhibited CaV2.1 currents. We have now determined the specificity of this effect, identified a potential mechanism, and have shown that such constructs also inhibit endogenous calcium currents when transfected into neuronal cell lines. Suppression of calcium channel expression requires interaction between truncated and full-length channels, because there is inter-subfamily specificity. Although there is marked cross-suppression within the CaV2 calcium channel family, there is no cross-suppression between CaV2 and CaV3 channels. The mechanism involves activation of a component of the unfolded protein response, the endoplasmic reticulum resident RNA-dependent kinase (PERK), because it is inhibited by expression of dominant-negative constructs of this kinase. Activation of PERK has been shown previously to cause translational arrest, which has the potential to result in a generalized effect on protein synthesis. In agreement with this, coexpression of the truncated domain I of CaV2.2, together with full-length CaV2.2, reduced the level not only of CaV2.2 protein but also the coexpressed α2δ-2. Thapsigargin, which globally activates the unfolded protein response, very markedly suppressed CaV2.2 currents and also reduced the expression level of both CaV2.2 and α2δ-2 protein. We propose that voltage-gated calcium channels represent a class of difficult-to-fold transmembrane proteins, in this case misfolding is induced by interaction with a truncated cognate CaV channel. This may represent a mechanism of pathology in episodic ataxia-2.

Interaction via a Key Tryptophan in the I-II Linker of N-Type Calcium Channels Is Required for β1 But Not for Palmitoylated β2, Implicating an Additional Binding Site in the Regulation of Channel Voltage-Dependent Properties

The CaVβ subunits of voltage-gated calcium channels regulate these channels in several ways. Here we investigate the role of these auxiliary subunits in the expression of functional N-type channels at the plasma membrane and in the modulation by G-protein-coupled receptors of this neuronal channel. To do so, we mutated tryptophan 391 to an alanine within the α-interacting domain (AID) in the I-II linker of CaV2.2. We showed that the mutation W391 virtually abolishes the binding of CaVβ1b and CaVβ2a to the CaV2.2 I-II linker and strongly reduced current density and cell surface expression of both CaV2.2/α2δ-2/β1b and/β2a channels. When associated with CaVβ1b, the W391A mutation also prevented the CaVβ1b-mediated hyperpolarization of CaV2.2 channel activation and steady-state inactivation. However, the mutated CaV2.2W391A/β1b channels were still inhibited to a similar extent by activation of the D2 dopamine receptor with the agonist quinpirole. Nevertheless, key hallmarks of G-protein modulation of N-type currents, such as slowed activation kinetics and prepulse facilitation, were not observed for the mutated channel. In contrast, CaVβ2a was still able to completely modulate the biophysical properties of CaV2.2W391A channel and allow voltage-dependent G-protein modulation of CaV2.2W391A. Additional data suggest that the concentration of CaVβ2a in the proximity of the channel is enhanced independently of its binding to the AID by its palmitoylation. This is essentially sufficient for all of the functional effects of CaVβ2a, which may occur via a second lower-affinity binding site, except trafficking the channel to the plasma membrane, which requires interaction with the AID region.

Variable number tandem repeat polymorphism of the mucin genes located in the complex on 11p15.5

Abstract A family of four genes that encode major secreted mucins (MUC6, MUC2, MUC5AC and MUC5B) map to within 400kb on chromosome 11p15.5. These genes contain long stretches of tandem repeats of sequence that encode serine- and threonine-rich domains but that otherwise show no similarity from gene to gene, and regions of unique sequence domains that do show evidence of sequence homology. We have previously reported the existence of polymorphism in three of these genes but the extent and nature of this allelic variation is now described here in detail. Variable number tandem repeat polymorphisms of MUC6, MUC2 and MUC5AC are predicted to encode mucin polypeptides that differ in length. In the case of MUC2 and MUC6 these length differences are substantial (up to twofold). MUC5B in contrast does not show common allele length variation. Three MUC2 mutations are reported, none of which are associated with the meiotic recombinations previously observed in this region of chromosome 11.

Genetic disruption of voltage-gated calcium channels in psychiatric and neurological disorders.

Highlights • Voltage-gated calcium channel classification—genes and proteins.• Genetic analysis of neuropsychiatric syndromes.• Calcium channel genes identified from GWA studies of psychiatric disorders.• Rare mutations in calcium channel genes in psychiatric disorders.• Pathophysiological sequelae of CACNA1C mutations and polymorphisms.• Monogenic disorders resulting from harmful mutations in other voltage-gated calcium channel genes.• Changes in calcium channel gene expression in disease.• Involvement of voltage-gated calcium channels in early brain development.

N Terminus Is Key to the Dominant Negative Suppression of CaV2 Calcium Channels: IMPLICATIONS FOR EPISODIC ATAXIA TYPE 2*

Expression of the calcium channels CaV2.1 and CaV2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in CaV2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both CaV2.1 and CaV2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg52 and Arg54) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of CaV2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in CaV2.2 protein level, and this is also prevented by mutation of Arg52 and Arg54 to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg52, Arg54 motif are involved in the processes underlying dominant negative suppression.Expression of the calcium channels Ca(V)2.1 and Ca(V)2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in Ca(V)2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both Ca(V)2.1 and Ca(V)2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg(52) and Arg(54)) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of Ca(V)2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in Ca(V)2.2 protein level, and this is also prevented by mutation of Arg(52) and Arg(54) to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg(52), Arg(54) motif are involved in the processes underlying dominant negative suppression.

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α2δ-1 subunits

Significance The auxiliary α2δ-1 subunits of voltage-gated calcium (CaV) channels are important therapeutic targets, representing the receptor for gabapentinoid drugs in neuropathic pain therapy. It is therefore important to understand their function. Because α2δ subunits augment calcium currents, it is believed that they increase cell-surface expression of these channels. Here, using exofacially tagged CaV2.2 constructs, we now show this to be the case. However, recent proteomic analysis found that α2δ subunits are associated only loosely and nonquantitatively with CaV2 channels, challenging their role as calcium channel subunits. In contrast, we find that CaV2.2 and α2δ-1 are intimately and completely associated at the plasma membrane and that this is not disrupted by the α2δ-1 ligand gabapentin, which reduces cell-surface expression of both CaV2.2 and α2δ-1. CaV1 and CaV2 voltage-gated calcium channels are associated with β and α2δ accessory subunits. However, examination of cell surface-associated CaV2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged CaV2 channels. Here we report the development of fully functional CaV2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the plasma membrane, in both a neuronal cell line and neurons. We first examined the effect of the auxiliary subunits. Although α2δ subunits copurify with CaV2 channels, it has recently been suggested that this interaction is easily disrupted and nonquantitative. We have now tested whether α2δ subunits are associated with these channels at the cell surface. We found that, whereas α2δ-1 is readily observed at the plasma membrane when expressed alone, it appears absent when coexpressed with CaV2.2/β1b, despite our finding that α2δ-1 increases plasma-membrane CaV2.2 expression. However, this was due to occlusion of the antigenic epitope by association with CaV2.2, as revealed by antigen retrieval; thus, our data provide evidence for a tight interaction between α2δ-1 and the α1 subunit at the plasma membrane. We further show that, although CaV2.2 cell-surface expression is reduced by gabapentin in the presence of wild-type α2δ-1 (but not a gabapentin-insensitive α2δ-1 mutant), the interaction between CaV2.2 and α2δ-1 is not disrupted by gabapentin. Altogether, these results demonstrate that CaV2.2 and α2δ-1 are intimately associated at the plasma membrane and allow us to infer a region of interaction.

DNA polymorphisms in the lactase gene. Linkage disequilibrium across the 70-kb region.

The enzyme lactase, which is responsible for the digestion of dietary lactose, is present in the intestine of some adults but not others. As a means of providing a platform to explore the molecular basis of this nutritionally relevant genetic variation we have screened for polymorphism in several regions of the lactase gene. In each case simple polymerase chain reaction-based procedures (including single-strand conformation analysis and denaturing gradient gel electrophoresis) were used, combined with silver staining as a method of detection. Allelic variation was found at 6 different sites. One previously published polymorphism was also tested. The frequencies of the alleles were determined in more than 100 unrelated individuals of the Centre d’Etude du Polymorphisme Humain (CEPH) panel, and the haplotypes were deduced. A region of linkage disequilibrium was observed, which spans the whole coding region of the lactase gene (∼ 60–70 kb); there were only 3 common haplotypes in this population. When the CEPH sample was subdivided according to the population of origin (France or Utah) the haplotype frequencies were shown to be markedly different.

Two additional polymorphisms within the hypervariable MUC1 gene: association of alleles either side of the VNTR region.

The gene MUC1 codes for a mucin‐type glycoprotein and like most of the other mucin genes shows variable number tandem repeat (VNTR) polymorphism within the coding region. A polymorphism due to a G/A substitution in exon 2, responsible for a genetically determined variation in splicing of the MUC1 transcript, has also been reported (Ligtenberg et al. 1990, 1991). Here we describe the detection of this nucleotide substitution polymorphism by single stranded conformational analysis of genomic DNA and we also report a CA repeat polymorphism within intron 6 of the gene. Haplotypes were determined in a series of families and the common alleles of these two polymorphisms were found to be associated. These results support the notion that the VNTR polymorphism in the coding sequence of MUC1 is not caused by unequal reciprocal recombination at meiosis.