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Editorial: Editorials

Interesting Insights from a Very Simple (Unicellular) Source

Lynch, Carl III, MD, PhD

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doi: 10.1213/ANE.0b013e3181c5b72b
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The nicotinic acetylcholine receptors (nAChRs), ionotropic γ-aminobutyric acid (GABA) receptors (GABAA and GABAC), glycine receptors, and ionotropic serotonin receptors (5HT3) all have a similar structure in which 5 membrane subunits of similar size and shape combine to form a transmembrane channel, something similar to the staves of a barrel. A large portion of each subunit extends out from the membrane into the extracellular fluid. This extracellular region contains a distinctive disulfide linkage, providing the “Cys-loop” moniker for these receptors.1 Based on the species distribution, this superfamily of pentameric ligand-gated ion channels (pLGICs) was thought to be restricted to multicellular organisms. However, recently, pentameric proteins with large extracellular ligand-binding domains have been described in single-celled organisms.2–5 Although these channels lack the Cys-loop, their overall structure is remarkably similar to the pLGICs (see Fig. 1). Importantly, as demonstrated in the report by Weng et al.,6 the proton-activated currents of these channels are sensitive to both volatile anesthetics and propofol. But why should anyone care about the effects of anesthetics on a membrane protein from a Cyanobacteria (blue-green algae) Gloeobacter (aka GLIC)?

Figure 1.
Figure 1.:
Structures of the Gloeobacter ligand-gated ion channel (GLIC, left) and a nicotinic acetylcholine receptor (nAChR, right) channel derived from radiograph diffraction and reproduced from the National Center for Biotechnology Information (code 3EHZ or 3EI0 for GLIC; code 2BG9 for nAChR; available at: http://www.ncbi.nlm.nih.gov/sites/entrez?db=domains). Each channel is composed of 5 subunits; 1 subunit in each channel is shown in cylinder (α-helix) and ribbon (β-sheet) format. The 5 M2 helices line the pore of the channel through which the ions pass. The M4 helix of each subunit actually extends most prominently into the lipid bilayer and is influenced by the order and structure of the lipid. The subunits of the AChR have an additional intracellular domain (MA) that forms a cage through which ions must pass. The linkage between the extracellular ligand-binding domain and the transmembrane bundle is mediated by salt bridges between these 2 regions (arrows). The approximate location of phospholipids and cholesterol, which constitute part of the bilayer, are shown in yellow.

Over the past 2 decades, by a combination of membrane electrophysiology, molecular biology, and structural proteomics (radiograph diffraction and nuclear magnetic resonance), we have come to a far more detailed understanding of the molecular basis of membrane excitation. Based on their primary structure/amino acid sequence, the multihelix bundles forming the structure of ion channels were proposed. Subsequent radiograph diffraction structural studies by MacKinnon7 and Unwin8,9 have defined the structure of ion channels at the atomic level and delineated the precise molecular rearrangements that occur as ion channels open and close. In Figure 1, the structure of GLIC deduced from radiograph diffraction data4,5 is shown next to the structure of an nAChR.8 Although the primary amino acid sequence shows a modest similarity of <20% with nAChR subunits, the similarity in secondary and tertiary structures is remarkable. Equally remarkable is the fact that when these channels are expressed in cells, they are inhibited by remarkably low concentrations of various anesthetic agents.

In considering the pLGICs it is noteworthy that although similar in structure, they have distinct behaviors. For example, the lining of transmembrane helices vary significantly such that nAChRs and 5HT3R transmit K+ and Na+ ions, whereas the GABA and glycine receptors transport Cl; the former depolarize the cell whereas the latter (usually) hyperpolarize or stabilize the membrane potential. More importantly, whereas anesthetics inhibit certain types of nAChR channels,10 anesthetics favor the opening of glycine and GABAA channels.11 For more than a decade, specific amino acid mutations in the nAChR12,13 and the GABAA14 receptor have been used to alter the sensitivity to volatile anesthetics and alcohol, and various investigators have inferred that specific binding sites exist on the channel protein. Are these differing anesthetic actions on various ion channels to be understood as actions of lipophilic molecules binding to distinct and randomly occurring amino acid sequences within each different membrane channel protein?

While we know that these pLGICs have a major role in mediating anesthetic actions, what can these bacterial channel members tell us? Despite divergent behavior and very modest homology in amino acid sequence, the similarity in overall structure of these pentameric proteins suggests that some common mechanisms of action may exist. Part of the problem in attempting to find a common theme is that in all these channels, the binding of a simple molecule to a cleft between the subunit extracellular regions results in significant movement of transmembrane helices so that there is large enough space for ions to pass through. The connection between the ligand-binding site and the transmembrane helices occurs over significant molecular distance (more than the thickness of the bilayer). Although yet to be completely elucidated, the change in structure seems to be mediated not by a fixed link but via electrostatic interactions (salt bridges) between the extracellular domain and transmembrane α-helices.15 This interaction site is at the plane of the membrane interfacial region—at the phospholipids head groups and polar portion of cholesterol. It is also in this region where tryptophan residues play an important role in anchoring the helical portion of the protein at the membrane surface.16 It seems more than coincidental that it is at the interfacial region of the membrane where volatile anesthetics reside.17,18

Another emerging clue is that many membrane proteins do not merely float in a sea of nonspecific lipid but instead have specific lipid requirements. For example, the function of the AChR is markedly enhanced and in some sense seems to require the presence of cholesterol in the bilayer,19,20 whereas various lipids can alter channel function21,22 or drug actions.23 Certain lipids are immobilized and remain tightly associated with pLGICs, while conversely membrane proteins also can cause specific organization of membrane lipids.24 Interestingly, the steroid hormone progesterone has anesthetic potency and similar to anesthetics inhibits AChR channels but activates GABAA channels. Anesthetically active steroids are large and bulky molecules, and it is difficult to imagine that it would occupy the same site as a small halogenated ether.

The view that anesthetics are acting at either a lipid or protein site may end up being far too simplistic. One suggestion is that the mechanical properties of the membrane lipids can mediate alterations in channel function.25–27 Although such global properties of the membrane may contribute, we are beginning to understand that there is a complex and sophisticated interaction between the specific lipids with various segments of the membrane proteins. For example, the “soft” interaction of the ligand-binding region with the transmembrane helices may require certain interfacial lipids. The linkage between the extracellular ligand-binding domain with the ion-gating region might be altered by a change in a critical amino acid in the region, a change in the lipid, or by anesthetic disruption of lipid-protein interaction. It is not impossible that lipids and anesthetics might weaken the linkage in one pLGIC (AChR) but strengthen it in another (GABA).

But what of these bacterial channels? The ability to mutate these molecules and produce them in sufficient quantity to study their function and their structure make them an exciting model for our more complex glycine, GABAA, nicotinic ACh, and 5HT3 channels. Photoactivated anesthetics may be used to determine with which regions of the receptor the drugs interact. Their protein structure can be mutated to determine the location of amino acids critical for permitting anesthetic action, and whether lipids will indeed have a role. Study of GLIC and ELIC3 (Erwinia chrysanthemi LIC, yet another bacterial LIC) will begin to answer how our structurally simple agents produce their panoply of actions.

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