Evidence for a common structure for a class of membrane channels (original) (raw)
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Structure and function of related proton channel-forming proteins
Pure and Applied Chemistry, 1994
A molecular model has been constructed for a 16 kDa integral membrane protein which is the principal component of gap junction-like structures in the arthropod Neplarops norvegicus. This proteolipid is a member of a family of highly conserved proteins comprising the proton channcl-forming subunits of vacuolar membrane (V-type) ATPases. The model suggests that the polypeptide exists as a transmembrane four-helical bundle, which assembles as a hexamer to form the membrane-spanning channel. The arthropod protein has been cloned and subsequently expressed in yeast, in which it complements a mutation in the endogenous gene for the related vacuolar membrane ATPase channel-forming subunit. Mutagenesis studies have been initiated in the yeast system in order to validate the structural model and to examine ion selectivity and transport mechanisms.
Distinct Structural Elements in the First Membrane-spanning Segment of the Epithelial Sodium Channel
Journal of Biological Chemistry, 2006
Epithelial Na ؉ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an ␣ 2 ␥ or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate site within ␣M1. We performed site-directed mutagenesis at this site and found that, although the mutants reduced channel activity, ENaC protein expression at the plasma membrane was unaffected. To deduce the role of ␣M1 in the pore structure of ENaC, we performed tryptophanscanning mutagenesis throughout ␣M1 (residues 110-130). We found that mutations within the amino-terminal part of ␣M1 had effects on activity and selectivity with a periodicity consistent with a helical structure but no effect on channel surface expression. We also observed that mutations within the carboxyl-terminal part of ␣M1 had effects on activity and selectivity but with no apparent periodicity. Additionally, these mutants reduced channel surface expression. Our data support a model in which the amino-terminal half of ␣M1 is ␣-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of ␣M1 may be helical or assume a different conformation and may be involved in tertiary interactions essential to proper channel folding or assembly. Together, our data suggest that ␣M1 is divided into two distinct regions.
The Origin and Early Evolution of Membrane Channels
Astrobiology, 2005
The origin and early evolution of ion channels are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly greater complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Bacteria, and Archaea). We discuss the potassiumsodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.
Biochemistry, 1999
The 16-kDa proteolipid from the hepatopancreas of Nephrops norVegicus belongs to the class of channel proteins that includes the proton-translocation subunit of the vacuolar ATPases. The membranous 16-kDa protein from Nephrops was covalently spin-labeled on the unique cysteine Cys54, with a nitroxyl maleimide, or on the functionally essential glutamate Glu140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). The intensities of the saturation transfer ESR spectra are a sensitive indicator of spin-spin interactions that were used to probe the intramembranous structure and assembly of the spinlabeled 16-kDa protein. Spin-lattice relaxation enhancements by aqueous Ni 2+ ions revealed that the spin label on Glu140 is located deeper within the membrane (around C9-C10 of the lipid chains) than is that on Cys54 (located around C5-C6). In double labeling experiments, alleviation of saturation by spin-spin interactions with spin-labeled lipids indicates that spin labels both on Cys54 and on Glu140 are at least partially exposed to the lipid chains. The decrease in saturation transfer ESR intensity observed with increasing spin-labeling level is evidence of oligomeric assembly of the 16-kDa monomers and is consistent with a protein hexamer. These results determine the locations and orientations of transmembrane segments 2 and 4 of the 16-kDa putative 4-helix bundle and put constraints on molecular models for the hexameric assembly in the membrane. In particular, the crucial DCCD-binding site that is essential for proton translocation appears to contact lipid.
Architecture of receptor-operated ion channels of biological membranes
Biophysics, 2011
Ion channels of biological membranes are the key proteins that provide for bioelectric functioning of living systems. These proteins are homo or heterooligomers assembled of several identical or different sub units. Understanding the architectural organization and functioning of ion channels has significantly expanded owing to resolving the crystal structure of several types of voltage gated and receptor operated channels. This review summarizes the information obtained from crystal structures of potassium channels, nicotinic acetylcholine receptor, ATP activated, and other ligand gated ion channels. Despite the differ ences in the function, topology, ion selectivity, and subunit stoichiometry, a high similarity in the principles of organization of these macromolecular complexes has been revealed.
Three-Dimensional Structure of a Recombinant Gap Junction Membrane Channel
Science, 1999
Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which is consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. The transmembrane alpha-helical rods contrasted with the double-layered appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu.