Despite the importance of Mg 2+ for numerous cellular activities, the mechanisms underlying its import and homeostasis are poorly understood. The CorA family is ubiquitous and is primarily responsible for Mg 2+ transport. However, the key questions-such as, the ion selectivity, the transport pathway, and the gating mechanism-have remained unanswered for this protein family. We present a 3.2 Å resolution structure of the archaeal CorA from Methanocaldococcus jannaschii, which is a unique complete structure of a CorA protein and reveals the organization of the selectivity filter, which is composed of the signature motif of this family. The structure reveals that polar residues facing the channel coordinate a partially hydrated Mg 2+ during the transport. Based on these findings, we propose a unique gating mechanism involving a helical turn upon the binding of Mg 2+ to the regulatory intracellular binding sites, and thus converting a polar ion passage into a narrow hydrophobic pore. Because the amino acids involved in the uptake, transport, and gating are all conserved within the entire CorA family, we believe this mechanism is general for the whole family including the eukaryotic homologs.

The CorA family of divalent cation transporters utilizes Mg2+ and Co2+ as primary substrates. The molecular mechanism of its function, including ion selectivity and gating, has not been fully characterized. Recently we reported a new structure of a CorA homologue from Methanocaldococcus jannaschii, which provided novel structural details that offered the conception of a unique gating mechanism involving conversion of an open hydrophilic gate into a closed hydrophobic one. In the present study we report functional evidence for this novel gating mechanism in the Thermotoga maritima CorA together with an improved crystal structure of this CorA to 2.7 Å (1 Å=0.1 nm) resolution. The latter reveals the organization of the selectivity filter to be similar to that of M. jannaschii CorA and also the previously unknown organization of the second signature motif of the CorA family. The proposed gating is achieved by a helical rotation upon the binding of a metal ion substrate to the regulatory binding sites. Additionally, our data suggest that the preference of this CorA for Co2+ over Mg2+ is controlled by the presence of threonine side chains in the channel. Finally, the roles of the intracellular metal-binding sites have been assigned to increased thermostability and regulation of the gating. These mechanisms most likely apply to the entire CorA family as they are regulated by the highly conserved amino acids.

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G-protein-coupled receptors (GPCRs) such as human muscarinic acetylcholine receptor 3 (hM3) assume dimeric/oligomeric forms in living cells while maintaining their ability to bind and activate G-proteins. The precise stoichiometry, quaternary organization, and stability of these receptor complexes in living cells remain a subject of significant controversy. We have investigated the organization of hM3 receptors in living cells using spectrally resolved two photon microscopy (SR-TPM). Wild type hM3 and a synthetic-ligand-regulated form of this receptor (RASSL), were expressed either constitutively or in an antibiotic dependent manner in Flp-InTM T-RExTM 293 cells [Alvarez-Curto et al, Journal of Biological Chemistry, 2010]. A so-called donor of energy (Cerulean Fluorescent Protein) fused to RASSL can get excited by laser light and transfers its energy to nearby (<10nm) acceptors (Yellow Fluorescent Protein) fused to wild type hM3 receptors through a non-radiative process called Forster Resonance Energy Transfer (FRET). Using our SR-TPM microscope, we were able to collect images showing distributions of single complexes of hM3-RASSL and hM3-WT formed at various levels of RASSL receptor expression and determined their apparent efficiency of energy transfer (Eapp). The calculated Eapp for individual complexes were binned according to their values in order to obtain a cumulative histogram of Eapp for all complexes. The experimental data thus obtained were fitted to theoretical models [Raicu et al, Nature Photonics, 2009] in order to determine the quaternary structure of the M3 receptor, which turned out to be a rhombus-shaped tetramer.

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