Background: The sigma-1 receptor modulates the activity of ion channels.Results: Atomic force microscopy imaging of complexes between sigma-1 receptors and Nav1.5 Na+ channels reveals a 4-fold symmetry.Conclusion: Each of the four sets of six transmembrane regions in Nav1.5 constitutes a sigma-1 receptor binding site.Significance: The sigma-1 receptor likely interacts with the transmembrane regions of its protein partners.

There is evidence that polycystin-2 (TRPP2) interacts with two other members of the transient receptor potential (TRP) family, TRPC1 and TRPV4. We have previously shown that TRPP2 forms a heteromeric complex with TRPC1, with a 2:2 stoichiometry and an alternating subunit arrangement. Here, we used coimmunoprecipitation to show that TRPP2 also interacts with TRPV4, but not with TRPA1 or TRPM8; hence, its promiscuity is limited. We then used atomic force microscopy to study the structure of the TRPV4 homomer and the interaction between TRPP2 and TRPV4. The molecular volume of V5-tagged TRPV4 isolated from singly-transfected tsA 201 cells indicated that it assembled as a homotetramer. The distribution of angles between pairs of anti-V5 antibodies bound to TRPV4 particles had a large peak close to 90 degrees and a smaller peak close to 180 degrees , again consistent with the assembly of TRPV4 as a homotetramer. In contrast, the angle distributions for decoration of the TRPP2-TRPV4 heteromer by either anti-Myc or anti-V5 antibodies had major peaks close to 180 degrees. This result indicates that TRPP2-TRPV4 assembles identically to TRPP2-TRPC1, suggesting a common subunit arrangement among heteromeric TRP channels.

The -1 receptor (Sig1R) is widely expressed in the CNS, where it has a neuroprotective role in ischemia and stroke and an involvement in schizophrenia. The Sig1R interacts functionally with a variety of ion channels, including the NMDA receptor (NMDAR). Here, we used atomic force microscopy (AFM) imaging to investigate the interaction between the Sig1R and the NMDAR. The Sig1R bound directly to GluN1/GluN2A NMDAR heterotetramers. Furthermore, the mean angle between pairs of bound Sig1Rs was 72°. This result suggested that the Sig1R interacts with either GluN1 or GluN2A, but not both, and supports our recent demonstration that the NMDAR subunits adopt an adjacent (i.e., 1/1/2/2) arrangement. The Sig1R could be coisolated with GluN1 but not with GluN2A, indicating that GluN1 is its specific target within the NMDAR. Consistent with this conclusion, AFM imaging of coisolated Sig1R and GluN1 revealed GluN1 dimers decorated with Sig1Rs. In situ proximity ligation assays demonstrated that the Sig1R interacts with GluN1 (but not with GluN2A) within intact cells and also that its C terminus is extracellular. We conclude that the Sig1R binds to the GluN1/GluN2A NMDAR specifically via the GluN1 subunit. This interaction likely accounts for at least some of the modulatory effects of Sig1R ligands on the NMDAR.

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (␣, ␤, and ␥); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the ␣-, ␤-and ␥-subunits. We show that for ␣-, ␤-and ␥-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that ␣␤␥-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits.The epithelial sodium channel (ENaC) 5 is a member of the ENaC/degenerin superfamily, which also includes acid-sensing ion channels (ASICs; reviewed in Ref. 1). ENaC mediates Na ϩ entry across the apical membranes of many absorptive epithelia, including the alveolar epithelium, salivary duct, distal colon, and sweat glands. ENaC plays a particularly important role in Na ϩ transport across the aldosterone-sensitive distal nephron (reviewed in Ref. 2), which is responsible for the regulation of Na ϩ and K ϩ excretion, and thus plays an important role in the control of blood pressure. ENaC is the target of potassiumsparing diuretics such as amiloride and spironolactone used in the treatment of cardiovascular disease. Whereas amiloride is a direct blocker of the channel, the mineralocorticoid receptor antagonist spironolactone inhibits ENaC activity by preventing its stimulation by aldosterone.ENaC is a heteromultimer containing three homologous subunits (␣, ␤, and ␥), which have 30 -40% amino acid identity (3, 4). A fourth ENaC subunit, ␦-ENaC, has been cloned from a human kidney library (5). In heterologous expression systems, ␦-ENaC has functional similarities with ␣-ENaC, but its physiological role is still unclear (6). Each ENaC subunit contains two transmembrane domains, a large extracellular loop, and short intracellular N and C termini. Full ENaC activity requires coexpression of all three subunits (4). In the past, it has been proposed that ENaC is assembled from either four (7-9) or eight to nine subunits (10, 11). However, the recently published crystal structure of ASIC 1a (12) suggests that ENaC is likely to be a trimer (13), although this has not been demonstrated.We have developed a method, based on AFM imaging, for determining the stoichiometry and arrangement of subunits within multimeric proteins (reviewed in Ref. 14). The method involves engineering specific epitope tags onto each protein subunit and expressing the proteins exogenously in a suitable cell line (usually tsA 201). Membrane fractions from the transfected cells are solubilized in detergent, and the proteins are isola...

BACKGROUND AND PURPOSEThe P2X receptor family consists of seven subunit types -P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits -P2X2 and P2X4, and P2X4 and P2X7. EXPERIMENTAL APPROACHWe used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging. KEY RESULTSBoth pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation. CONCLUSIONS AND IMPLICATIONSWe conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.