Andrew P. Stewart

The σ-1 Receptor Interacts Directly with GluN1 But Not GluN2A in the GluN1/GluN2A NMDA Receptor

The σ-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.

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (α, β, and γ); 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 α-, β- and γ-subunits. We show that for α-, β- and γ-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 αβγ-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.

Atomic Force Microscopy Reveals the Alternating Subunit Arrangement of the TRPP2-TRPV4 Heterotetramer

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 Sigma-1 Receptor Binds to the Nav1.5 Voltage-gated Na+ Channel with 4-Fold Symmetry

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. The sigma-1 receptor (Sig1R) is up-regulated in many human tumors and plays a role in the control of cancer cell proliferation and invasiveness. At the molecular level, the Sig1R modulates the activity of various ion channels, apparently through a direct interaction. We have previously shown using atomic force microscopy imaging that the Sig1R binds to the trimeric acid-sensing ion channel 1A with 3-fold symmetry. Here, we investigated the interaction between the Sig1R and the Nav1.5 voltage-gated Na+ channel, which has also been implicated in promoting the invasiveness of cancer cells. We show that the Sig1R and Nav1.5 can be co-isolated from co-transfected cells, consistent with an intimate association between the two proteins. Atomic force microscopy imaging of the co-isolated proteins revealed complexes in which Nav1.5 was decorated by Sig1Rs. Frequency distributions of angles between pairs of bound Sig1Rs had two peaks, at ∼90° and ∼180°, and the 90° peak was about twice the size of the 180° peak. These results demonstrate that the Sig1R binds to Nav1.5 with 4-fold symmetry. Hence, each set of six transmembrane regions in Nav1.5 likely constitutes a Sig1R binding site, suggesting that the Sig1R interacts with the transmembrane regions of its partners. Interestingly, two known Sig1R ligands, haloperidol and (+)-pentazocine, disrupted the Nav1.5/Sig1R interaction both in vitro and in living cells. Finally, we show that endogenously expressed Sig1R and Nav1.5 also functionally interact.

P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers

BACKGROUND AND PURPOSE The 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.

Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging

There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica. We detected a mixture of subunit monomers, dimers and trimers. In some cases, triple-subunit clusters were clearly visible, confirming the trimeric structure of the channel, and indicating that the trimer sometimes disaggregated after adhesion to the mica surface. This AFM-based technique will now enable us to determine the subunit arrangement within heteromeric ASICs.

The Kv7.2/Kv7.3 heterotetramer assembles with a random subunit arrangement

Background: Kv7.2 and Kv7.3 coassemble to form a heteromeric K+ channel that generates the M-current. Results: Atomic force microscopy (AFM) imaging of Kv7.2/Kv7.3 heteromers in complex with subunit-specific antibodies reveals the channel architecture. Conclusion: The Kv7.2/Kv7.3 heteromer assembles with a random subunit arrangement. Significance: Unusually, heteromeric Kv7 channels have a variable architecture. Voltage-gated K+ channels composed of Kv7.2 and Kv7.3 are the predominant contributors to the M-current, which plays a key role in controlling neuronal activity. Various lines of evidence have indicated that Kv7.2 and Kv7.3 form a heteromeric channel. However, the subunit stoichiometry and arrangement within this putative heteromer are so far unknown. Here, we have addressed this question using atomic force microscopy imaging of complexes between isolated Kv7.2/Kv7.3 channels and antibodies to epitope tags on the two subunits, Myc on Kv7.2 and HA on Kv7.3. Initially, tsA 201 cells were transiently transfected with equal amounts of cDNA for the two subunits. The heteromer was isolated through binding of either tag to immunoaffinity beads and then decorated with antibodies to the other tag. In both cases, the distribution of angles between pairs of bound antibodies had two peaks, at around 90° and around 180°, and in both cases the 90° peak was about double the size of the 180° peak. These results indicate that the Kv7.2/Kv7.3 heteromer generated by cells expressing approximately equal amounts of the two subunits assembles as a tetramer with a predominantly 2:2 subunit stoichiometry and with a random subunit arrangement. When the DNA ratio for the two subunits was varied, copurification experiments indicated that the subunit stoichiometry was variable and not fixed at 2:2. Hence, there are no constraints on either the subunit stoichiometry or the subunit arrangement.

α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N-Methyl-d-aspartate (NMDA) Receptors Adopt Different Subunit Arrangements

Background: Recent evidence has suggested that the various ionotropic glutamate receptors are assembled in the same way from their constituent subunits. Results: Atomic force microscopy imaging of antibody-decorated AMPA and NMDA receptors revealed different subunit arrangements. Conclusion: AMPA receptors have an alternating subunit arrangement, whereas NMDA receptors adopt an adjacent arrangement. Significance: Our results necessitate a reassessment of ionotropic glutamate receptor assembly. Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. All three ionotropic glutamate subfamilies (i.e. AMPA-type, kainate-type, and NMDA-type) assemble as tetramers of four homologous subunits. There is good evidence that both heteromeric AMPA and kainate receptors have a 2:2 subunit stoichiometry and an alternating subunit arrangement. Recent studies based on presumed structural homology have indicated that NMDA receptors adopt the same arrangement. Here, we use atomic force microscopy imaging of receptor-antibody complexes to show that whereas the GluA1/GluA2 AMPA receptor assembles with an alternating (i.e. 1/2/1/2) subunit arrangement, the GluN1/GluN2A NMDA receptor adopts an adjacent (i.e. 1/1/2/2) arrangement. We conclude that the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. This surprising finding necessitates a reassessment of the assembly of these important receptors.

Identification of free cytoplasmic globin mRNA of duck erythroblasts by hybridization to anti-messenger DNA and by cell-free protein synthesis.

The cytoplasm of animal cells contains mRNA not associated with ribosomes which exists in the form of ribonucleoprotein (RNP) complexes [ 1,2] . It has been suggested that such complexes represent a pool of mRNA intermediate between the nuclear pre-mRNA and the mRNP complex active in translation [l-4] . We have described in immature duck erythrocytes a cytoplasmic 9 S RNA not associated with ribosomes. This free cytoplasmic 9 S RNA has the same electrophoretic mobility as polyribosoma19 S globin mRNA; its rate of synthesis and decay is consistent with, but not a proof of, a role as a precursor to polyribosomal 9 S mRNA [4] . In this paper we present direct proof for the existence of a cytoplasmic pool of genuine globin mRNA free from the ribosomes (free mRNA) by demonstrating the capacity of the free 9 S RNA to be translated into globins and to hybridize with anti-messenger DNA (amDNA) copied from globin mRNA by reverse transcriptase.

Atomic force microscopy imaging reveals the domain structure of polycystin-1.

Mutation of polycystin-1 (PC1) is the major cause of autosomal dominant polycystic kidney disease. PC1 has a predicted molecular mass of ~460 kDa comprising a long multidomain extracellular N-terminal region, 11 transmembrane regions, and a short C-terminal region. Because of its size, PC1 has proven difficult to handle biochemically, and structural information is consequently sparse. Here we have isolated wild-type PC1, and several mutants, from transfected cells by immunoaffinity chromatography and visualized individual molecules using atomic force microscopy (AFM) imaging. Full-length PC1 appeared as two unequally sized blobs connected by a 35 nm string. The relative sizes of the two blobs suggested that the smaller one represents the N-terminus, including the leucine-rich repeats, the first polycystic kidney disease (PKD) domain, and the C-type lectin motif, while the larger one is the C-terminus, including the receptor for egg jelly (REJ) domain, all transmembrane domains, and the cytoplasmic tail. The intervening string would then consist of a series of tandem PKD domains. The structures of the various PC1 mutants were all consistent with this model. Our results represent the first direct visualization of the structure of PC1, and reveal the architecture of the protein, with intriguing implications for its function.