The R-Domain: Identification of an N-terminal Region of the α2δ-1 Subunit Which is Necessary and Sufficient for its Effects on Cav2.2 Calcium Currents.

Voltage-gated calcium channels (Cav) and their associated proteins are pivotal signalling complexes in excitable cell physiology. In nerves and muscle, Cav tailor calcium influx to processes including neurotransmission, muscle contraction and gene expression. Cav comprise a pore-forming α1 and modulatory ß and α2δ subunits - the latter targeted by anti-epileptic and anti-nociceptive gabapentinoid drugs. However, the mechanisms of gabapentinoid action are unclear, not least because detailed structure-function mapping of the α2δ subunit remains lacking. Using molecular biology and electrophysiological approaches we have conducted the first systematic mapping of α2δ subunit structure-function. We generated a series of cDNA constructs encoding chimera, from which successive amino acids from the rat α2δ-1 subunit were incorporated into a Type 1 reporter protein - PIN-G, to produce sequential extensions from the transmembrane (TM) region towards the N-terminus. By successive insertion of a TGA stop codon, a further series of N- to C-terminal extension constructs lacking the TM region, were also generated. Using this approach we have defined the minimal region of α2δ-1 - we term the R-domain (Rd), that appears to contain all the machinery necessary to support the electrophysiological and trafficking effects of α2δ-1 on Cav. Structural algorithms predict that Rd is conserved across all four α2δ subunits, including RNA splice variants, and irrespective of phyla and taxa. We suggest, therefore, that Rd likely constitutes the major locus for physical interaction with the α1 subunit and may provide a target for novel Cav therapeutics.

Insights into desmosome biology from inherited human skin disease and cardiocutaneous syndromes.

The importance of desmosomes in tissue homeostasis is highlighted by natural and engineered mutations in desmosomal genes, which compromise the skin or heart and in some instances both. Desmosomal gene mutations account for 45-50% of cases of arrhythmogenic right ventricular cardiomyopathy, and are mutated in an array of other disorders such as striate palmoplantar keratoderma, hypotrichosis with or without skin vesicles and lethal acantholytic epidermolysis bullosa. Recently, we reported loss-of-function mutations in the human ADAM17 gene, encoding for the 'sheddase' ADAM17, a transmembrane protein which cleaves extracellular domains of substrate proteins including TNF-α, growth factors and desmoglein (DSG) 2. Patients present with cardiomyopathy and an inflammatory skin and bowel syndrome with defective DSG processing. In contrast, the dominantly inherited tylosis with oesophageal cancer appears to result from gain-of-function in ADAM17 due to increased processing via iRHOM2. This review discusses the heterogeneity of mutations in desmosomes and their regulatory proteins.

iRHOM2-dependent regulation of ADAM17 in cutaneous disease and epidermal barrier function.

iRHOM2 is a highly conserved, catalytically inactive member of the Rhomboid family, which has recently been shown to regulate the maturation of the multi-substrate ectodomain sheddase enzyme ADAM17 (TACE) in macrophages. Dominant iRHOM2 mutations are the cause of the inherited cutaneous and oesophageal cancer-susceptibility syndrome tylosis with oesophageal cancer (TOC), suggesting a role for this protein in epithelial cells. Here, using tissues derived from TOC patients, we demonstrate that TOC-associated mutations in iRHOM2 cause an increase in the maturation and activity of ADAM17 in epidermal keratinocytes, resulting in significantly upregulated shedding of ADAM17 substrates, including EGF-family growth factors and pro-inflammatory cytokines. This activity is accompanied by increased EGFR activity, increased desmosome processing and the presence of immature epidermal desmosomes, upregulated epidermal transglutaminase activity and heightened resistance to Staphylococcal infection in TOC keratinocytes. Many of these features are consistent with the presence of a constitutive wound-healing-like phenotype in TOC epidermis, which may shed light on a novel pathway in skin repair, regeneration and inflammation.

Rhomboid proteins: a role in keratinocyte proliferation and cancer.

The Rhomboids represent a relatively recently discovered family of proteins, consisting in a variety of intramembrane serine proteases and their inactive homologues, the iRhoms. Rhomboids typically contain six or seven transmembrane domains (TMD) and have been classified into four subgroups: Secretase A and B, Presenilin-Associated-Rhomboid-Like (PARL) and iRhoms. Although the iRhoms, iRhom1 and iRhom2, have lost their protease activity during evolution, they retain key non-protease functions and have been implicated in the regulation of epidermal growth factor (EGF) signalling. EGF is moreover a substrate of RHBDL2, their active Rhomboid relative. Other substrates of RHBDL2 include members of the EphrinB family and thrombomodulin. RHBDL2 has also previously been demonstrated to be important in wound healing in cutaneous keratinocytes through the cleavage of thrombomodulin. Additional roles for these intriguing proteins seem likely to be revealed in the future. This review focuses on our current understanding of Rhomboids and, in particular, on RHBDL2 and iRhom2 and their roles in cellular processes and human disease.

RHBDF2 mutations are associated with tylosis, a familial esophageal cancer syndrome.

Tylosis esophageal cancer (TOC) is an autosomal-dominant syndrome characterized by palmoplantar keratoderma, oral precursor lesions, and a high lifetime risk of esophageal cancer. We have previously localized the TOC locus to a small genomic interval within chromosomal region 17q25. Using a targeted capture array and next-generation sequencing, we have now identified missense mutations (c.557T>C [p.Ile186Thr] and c.566C>T [p.Pro189Leu] in RHBDF2, which encodes the inactive rhomboid protease RHBDF2 (also known as iRhom2), as the underlying cause of TOC. We show that the distribution of RHBDF2 in tylotic skin is altered in comparison with that in normal skin, and immortalized tylotic keratinocytes have decreased levels of total epidermal growth factor receptor (EGFR) and display an increased proliferative and migratory potential relative to normal cells, even when normal cells are stimulated with exogenous epidermal growth factor. It would thus appear that EGFR signaling is dysregulated in tylotic cells. Furthermore, we also show an altered localization of RHBDF2 in both tylotic and sporadic squamous esophageal tumors. The elucidation of a role of RHBDF2 in growth-factor signaling in esophageal cancer will help to determine whether targeting this pathway in chemotherapy for this and other squamous cell carcinomas will be effective.

Targeting of voltage-gated calcium channel α2δ-1 subunit to lipid rafts is independent from a GPI-anchoring motif.

Voltage-gated calcium channels (Ca(v)) exist as heteromultimers comprising a pore-forming α(1) with accessory ß and α(2)δ subunits which modify channel trafficking and function. We previously showed that α(2)δ-1 (and likely the other mammalian α(2)δ isoforms--α(2)δ-2, 3 and 4) is required for targeting Ca(v)s to lipid rafts, although the mechanism remains unclear. Whilst originally understood to have a classical type I transmembrane (TM) topology, recent evidence suggests the α(2)δ subunit contains a glycosylphosphatidylinositol (GPI)-anchor that mediates its association with lipid rafts. To test this notion, we have used a strategy based on the expression of chimera, where the reported GPI-anchoring sequences in the gabapentinoid-sensitive α(2)δ-1 subunit have been substituted with those of a functionally inert Type I TM-spanning protein--PIN-G. Using imaging, electrophysiology and biochemistry, we find that lipid raft association of PIN-α(2)δ is unaffected by substitution of the GPI motif with the TM domain of PIN-G. Moreover, the presence of the GPI motif alone is not sufficient for raft localisation, suggesting that upstream residues are required. GPI-anchoring is susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage. However, whilst raft localisation of PIN-α(2)δ is disrupted by PI-PLC treatment, this is assay-dependent and non-specific effects of PI-PLC are observed on the distribution of the endogenous raft marker, caveolin, but not flotillin. Taken together, these data are most consistent with a model where α(2)δ-1 retains its type I transmembrane topology and its targeting to lipid rafts is governed by sequences upstream of the putative GPI anchor, that promote protein-protein, rather than lipid-lipid interactions.

Formation of N-type (Cav2.2) voltage-gated calcium channel membrane microdomains: Lipid raft association and clustering.

Voltage-gated calcium channels (Ca(v)s) comprise a pore-forming α1 with auxiliary α2δ and ß subunits which modulate Ca(v) function and surface expression. Ca(v)α1 and α2δ are present in signalling complexes termed lipid rafts but it is unclear whether α2δ is obligatory for targeting Ca(v)s to rafts or to what extent this influences cell surface organisation of Ca(v)s. Here, we have used imaging, biochemistry and electrophysiology to determine localisation and raft-partitioning of WT and functionally active HA-epitope tagged α2δ-1 and Ca(v)2.2 subunits expressed in COS-7 cells. We show that α2δ-1 not only partitions into lipid rafts itself but also mediates raft-partitioning of Ca(v)2.2/ß(1b) complexes. Ca(v)α2δ-1, Ca(v)2.2/ß(1b) and Ca(v)2.2/ß(1b)/α2δ-1 complexes are all organised into cell surface clusters although only in the presence of α2δ-1 do they co-localise with raft markers, caveolin and flotillin. Such clusters persist in the presence of 3-methyl-ß-cyclodextrin even though the raft markers disperse. However, clustering is profoundly sensitive to disruption of the actin-based cytoskeleton by cytochalasin-D. We conclude that α2δ-1, and likely other α2δ subunits, is necessary and sufficient for targeting Ca(v)s to lipid rafts. However, formation of clusters supporting "hotspots" of Ca(v) activity requires aggregation of macromolecular complexes containing raft components, stabilised by interactions with the cytoskeleton.

Organ culture mimics the effects of hypoxia on membrane potential, K(+) channels and vessel tone in pulmonary artery.

BACKGROUND AND PURPOSE: Blood vessel culture is gaining interest for use with transfection-based techniques, but alters the contractile properties of the vessels. The present study tested the effects of culture on the intrinsic tone of rat pulmonary arteries (PAs) and examined the function and expression of K(+) channels regulating the resting membrane potential (E(m)) and tone of pulmonary artery smooth muscle cells (PASMCs). EXPERIMENTAL APPROACH: Rat intrapulmonary arteries were isolated and cultured under standard and modified conditions. Contractile responses of fresh and cultured PA were compared using vessel myograph. Electrophysiology experiments on isolated PASMCs used the patch-clamp technique. K(+) channel expression was quantified using reverse transcription and real-time PCR. KEY RESULTS: After 4 days in culture vessels contracted to phenylephrine, but relaxation to carbachol was significantly impaired. Contractile responses to 10 mM KCl, 4-aminopyridine and tetraethylammonium increased, and vessels developed an uncharacteristic relaxation response to Ca(2+)-free solution, nifedipine and levcromakalim. PASMCs from cultured vessels were depolarized and K(+) currents reduced, in association with down-regulation of K(v)1.5, K(v)2.1 and TWIK-related acid-sensitive K(+) channel-1 mRNA. These changes were partially reversed by increased oxygenation of the culture medium or removing the endothelium before culture. CONCLUSIONS AND IMPLICATIONS: Culture of PA for 3-4 days induced loss of functional K(+) channels, depolarization of PASMCs, Ca(2+) influx, intrinsic tone and spontaneous constrictions, similar to the effects of chronic hypoxia. This limits the use of cultured vessels for studying excitation-contraction coupling, although oxygenating the culture medium and removing the endothelium can help to retain normal smooth muscle function.