We examined functional consequences of intrasubunit contacts in the nicotinic receptor ␣ subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues ␣ K145, ␣ D200, and ␣ Y190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, primarily by slowing the rate of channel opening. The combined mutations ␣ D200N and ␣ K145Q impair channel gating to the same extent as either single mutation, while ␣ K145E counteracts the impaired gating due to ␣ D200K, further suggesting electrostatic interaction between these residues. Interpreted in light of the crystal structure of acetylcholine binding protein (AChBP) with bound carbamylcholine (CCh), the results suggest in the absence of ACh, ␣ K145 and ␣ D200 form a salt bridge associated with the closed state of the channel. When ACh binds, ␣ Y190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches ␣ K145, which in turn loosens its contact with ␣ D200. The positional changes of ␣ K145 and ␣ D200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of ␤ -strand 7, which harbors ␣ K145 and is part of the signature Cys-loop, and the second is of ␤ -strand 10, which harbors ␣ D200 and connects to the M1 domain. Thus, interplay between these three conserved residues relays the initial conformational change from the ACh binding site toward the ion channel.

Potentiation of neuronal nicotinic acetylcholine receptors by exogenous ligands is a promising strategy for treatment of neurological disorders including Alzheimer's disease and Schizophrenia. To gain insight into molecular mechanisms underlying potentiation, we examined ACh-induced single-channel currents through the human neuronal α7 acetylcholine receptor in the presence of the α7-specific potentiator PNU-120596 (PNU). Compared to the unusually brief single-channel opening episodes elicited by agonist alone, channel opening episodes in the presence of agonist and PNU are dramatically prolonged. Dwell time analysis reveals that PNU introduces two novel components into open-time histograms, indicating at least two degrees of PNU-induced potentiation. Openings of the longest potentiated class coalesce into clusters whose frequency and duration change over a narrow range of PNU concentration. At PNU concentrations approaching saturation, these clusters last up to several minutes, prolonging the sub-millisecond α7 opening episodes by several orders of magnitude. Mutations known to reduce PNU potentiation at the whole-cell level still give rise to multi-second long single-channel clusters. However mutation of five residues lining a cavity within each subunit's transmembrane domain abolishes PNU potentiation, defining minimal structural determinants of PNU potentiation.

The nicotinic acetylcholine receptor (AChR) transduces binding of nerve-released ACh into opening of an intrinsic ion channel, yet the intraprotein interactions behind transduction remain to be fully elucidated. Attention has focused on the region of the AChR in which the ␤1-␤2 and Cys-loops from the extracellular domain project into a cavity framed by residues preceding the first transmembrane domain (pre-M1) and the linker spanning transmembrane domains M2 and M3. Previous studies identified a principal transduction pathway in which the pre-M1 domain is coupled to the M2-M3 linker through the ␤1-␤2 loop. Here we identify a parallel pathway in which the pre-M1 domain is coupled to the M2-M3 linker through the Cys-loop. Mutagenesis, single-channel kinetic analyses and thermodynamic mutant cycle analyses reveal energetic coupling among ␣Leu 210 from the pre-M1 domain, ␣Phe 135 and ␣Phe 137 from the Cys-loop, and ␣Leu 273 from the M2-M3 linker. Residues at equivalent positions of non-␣-subunits show negligible coupling, indicating these interresidue couplings are specific to residues in the ␣-subunit. Thus, the extracellular ␤1-␤2 and Cys-loops bridge the pre-M1 domain and M2-M3 linker to transduce agonist binding into channel gating.

Nicotinic acetylcholine receptors (AChRs) mediate rapid excitatory synaptic transmission throughout the peripheral and central nervous systems. They transduce binding of nerve-released ACh into opening of an intrinsic channel, yet the structural basis underlying transduction is not fully understood. Previous studies revealed a principal transduction pathway in which αArg 209 of the pre-M1 domain and αGlu 45 of the β1–β2 loop functionally link the two regions, positioning αVal 46 of the β1–β2 loop in a cavity formed by αPro 272 through αSer 269 of the M2–M3 loop. Here we investigate contributions of residues within and proximal to this pathway using single-channel kinetic analysis, site-directed mutagenesis, and thermodynamic mutant cycle analysis. We find that in contributing to channel gating, αVal 46 and αVal 132 of the signature Cys loop couple energetically to αPro 272. Furthermore, these residues are optimized in both their size and hydrophobicity to mediate rapid and efficient channel gating, suggesting naturally occurring substitutions at these positions enable a diverse range of gating rate constants among the Cys-loop receptor superfamily. The overall results indicate that αPro 272 functionally couples to flanking Val residues extending from the β1–β2 and Cys loops within the ACh binding to channel opening transduction pathway.

α-Bungarotoxin (α-Btx) binds to the five agonist binding sites on the homo-pentameric α7 acetylcholine receptor, yet the number of bound α-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for α-Btx blockade, we generate receptors comprised of wild-type and α-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with α-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by α-Btx and accessible to the agonist. Given structural evidence that α-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five α7 subunits are interdependent and maintain conformational symmetry in the open channel state.

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DC-SIGN, a dendritic cell (DC)-specific C-type lectin that binds many different pathogens, is a receptor for HIV-1 and promotes subsequent infection of T cells. Our previous study demonstrated that DC-SIGN forms sub-micron scale domains on the surface of immature DCs as well as cell lines that ectopically express DC-SIGN. In this study, we investigated the occupancy and dynamics of DC-SIGN surface domains. First, we developed a single molecule approach, based on total internal reflection fluorescence (TIRF) microscopy, to examine the number of DC-SIGN molecules in a single domain. By comparing the brightness of a single fluorophore to the total brightness of a fluorescently-labeled DC-SIGN domain, we show that the number of DC-SIGN proteins in a domain ranges from a few to over hundred molecules. The size of each domain, as measured from the full-width half-maximum (FWHM) of a Gaussian fit of the emission profile of a domain, varies from the diffraction limit to micron scale. Second, scanning fluorescence correlation spectroscopy (sFCS) and fluorescence recovery after photobleaching (FRAP) were carried out to investigate the mobility of DC-SIGN in a domain, both of which showed that DC-SIGN is highly immobile. This was corroborated by single particle tracking of quantum dots attached to DC-SIGN molecules within a domain. By contrast, photobleaching of a lipid modified-fluorescent protein (mRFP) in a DC-SIGN domain area showed full recovery similar to that outside of the domain, indicating that lipids inside the DC-SIGN domain are highly mobile and and can freely exchange with the surrounding membrane. Finally, a deletion mutation study of DC-SIGN was carried out to further investigate which moiety of DC-SIGN facilitates surface domain formation. Supported by NIH GM41402.