Intramembranous Segment Research Articles

Structure of the sodium channel pore revealed by serial cysteine mutagenesis.

The pores of voltage-gated cation channels are formed by four intramembrane segments that impart selectivity and conductance. Remarkably little is known about the higher order structure of these critical pore-lining or P segments. Serial cysteine mutagenesis reveals a pattern of side-chain accessibility that contradicts currently favored structural models based on alpha-helices or beta-strands. Like the active sites of many enzymes of known structure, the sodium channel pore consists of irregular loop regions.

The transferrin receptor and the release of iron from transferrin.

In a classic report detailing the role of transferrin in providing iron to human reticulocytes, Jand1 and coworkers noted that uptake of transferrin-borne iron was saturable as the concentration of transferrin in the culture medium increased, and that uptake was essentially abolished if cells were first exposed to trypsin. 1 On the basis of these observations, the existence of a cell-surface receptor for transferrin was first postulated. Numerous attempts to solubilize, isolate and characterize the transferrin receptor inevitably followed, but at first with little concordance in reported findings. Ultimately, however, such efforts led to the recognition that the transferrin receptor was a disulfide-linked homodimer of 95,000 KDa glycosylated subunits’2-4 still capable of binding transferrin when solubilized in non-denaturing detergent. 5 Cloning of receptor then definitively established that its cDNA coded for an amino acid sequence of 760 residues with a calculated molecular weight of 84, 910. 6 The two chains comprising the intact receptor are joined by a pair of disulfide bonds within the intramembranous portion of the receptor, with anchoring to the membrane facilitated by a covalently-bound fatty acid residue. 7 Hydropathy analysis suggests that this intramembranous segment consists of 28 amino acids (residues 62 to 89 in the sequence); the remainder of the N-terminal sequence would then lie within the cytoplasm. The bulk of the C-terminal region bearing the transferrin-binding site is disposed extracellularly, accessible to the extracellular space. N- and O-linked oligosaccharides8,9 may serve to maintain the extracellular domain in free contact with the bathing fluid, and thereby poised to capture transferrin.

Acetylcholine receptor synthesis rate and levels of receptor subunit messenger RNAs in chick muscle

Levels of mRNAs specific for the α-, γ- and δ-subunit of the nicotinic acetylcholine receptor were measured in chick skeletal muscle by solution hybridization, using a genomic DNA probe containing the intramembrane segments M2 and M3 of the α-subunit 2 and probes comprising exons 2–6 and exons 4–8, respectively, of the γ- and δ-subunit. 27 In the innervated calf musculature of adult chickens, receptor-specific messages were detected in approx. 100-fold excess over the amount required to account for the observed synthesis rate. Within 1 week after section of the sciatic nerve, α-, γ- and δ-subunit message levels rose 112-, 42- and 24-fold, respectively, while receptor expression rate increased about 150-fold. The rise in message levels preceded the denervation-induced increase in receptor concentration. In differentiating myogenic cells all three messages were found in excess over the amounts required for the observed rate of receptor synthesis. Treatment of differentiated myotubes with drugs that change receptor synthesis rate selectively affects α-subunit mRNA. In all situations in vitro and in vivo the α-subunit mRNA was found to reach final levels faster, and to be from 3 to over 30 times more abundant, than the other messages. These observations corroborate earlier evidence for a regulatory mechanism in which the supply of mRNA determines acetylcholine receptor synthesis rate. They also suggest that receptor expression is not simply proportional to acetylcholine receptor subunit mRNA concentrations, but rather is controlled, to a considerable extent, by the efficiency with which the receptor-specific mRNAs and/or the subunits they code for are subsequently utilized.

Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study.

A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.

Isolation and characterization of the hydrophobic COOH-terminal domains of the Sindbis virion glycoproteins

Digestion of intact Sindbis virions with α-chymotrypsin produced a single membrane-associated peptide derived from each of the two virion glycoproteins (referred to as RE1 and RE2, or roots derived from E1 and E2, respectively). Amino acid composition data and NH 2-terminal sequence analysis established their location at the extreme COOH-terminal end of each glycoprotein. RE1 and RE2 are rich in hydrophobia amino acids and insoluble in aqueous solutions in the absence of detergents, and show differential solubility in organic solvent systems designed for the extraction of lipids. Essentially all of the covalently attached palmitic acid associated with E1 and E2 was found to be clustered in their hydrophobic, membrane-associated roots. Beginning six to seven residues from their NH 2 termini, RE1 and RE2 contain uninterrupted sequences of hydrophobic amino acids similar in terms of amino acid composition and length to the transmembrane anchors found in other bitopic integral membrane proteins. By comparing the sequence and composition data obtained here with the sequences of E1 and E2 deduced from complementary DNA sequence analysis (Rice & Strauss, 1981) we can make several observations. First, following their uncharged, putative intramembrane segments (33 and 26 amino acids, respectively), E1 and E2 contain clusters of predominantly basic amino acids. By structural analogy to known transmembrane proteins, E1 probably spans the bilayer but contains only a few residues exposed on the inner face of the virion envelope. In contrast, E2 and PE2 (the precursor to E2), which have been shown to span the bilayer completely, contain an additional 33 COOH-terminal residues, which could be either exposed on the cytoplasmic face of the lipid bilayer or which could loop back into the membrane. This region at the extreme COOH-terminal end of E2, which is protected by the virion envelope from digestion by a-chymotrypsin, contains a second uncharged domain (23 amino acids in length) whose orientation is unknown, but which may be involved in the highly specific interaction of the transmembrane glycoproteins in the plasma membrane with the cytoplasmic nucleocapsid during budding.

Amino acid sequence of the carboxyl-terminal hydrophilic region of the H-2Kb MHC alloantigen. Completion of the entire primary structure of the H-2Kb molecule.

The amino acid sequence of the COOH-terminal hydrophilic region of the H-2Kb histocompatibility antigen was determined. The sequence was completed by analyses of four CNBr fragments obtained from the intact molecule as well as tryptic peptides. This region was composed of 39 amino acid residues with a cluster of basic residues at the NH2 terminus and localized positions 308-346 of the H-2Kb molecule. These sequence data, together with those reported for the NH2-terminal 284 residues [Martinko, J. M., Uehara, H., Ewenstein, B. M., Kindt, T. J., Coligan, J. E., & Nathenson, S. G. (1980) Biochemistry 19, 6188-6193] and for the intramembranous segment [Uehara, H., Coligan, J. E., & Nathenson, S. G. (1981) Biochemistry (preceding paper in this issue)], provided the complete primary structure of the H-2Kb molecule. This is the first histocompatibility antigen for which the entire primary structure is determined.

Isolation and sequence analysis of the intramembranous hydrophobic segment of the H-2Kb murine histocompatibility antigen.

The primary structure of the intramembranous segment (TC-1) of the mouse transplantation antigen, H-2Kb, has been determined. The segment contains a stretch of 31 uncharged amino acid residues and is localized between the NH2-terminal and the COOH-terminal hydrophilic regions of the molecule. The amino acid sequence of TC-1 is Trp-Asp-Glu-Pro-Pro-Ser-Thr-Val-Ser-Asn-Met-Ala-Thr-Val-Ala-Val-Leu-Val-Val-Leu -Gly-Ala-Ala-Ile-Val-Thr-Gly-Ala-Val-Val-Ala-Phe-Val-Met-Lys-Met-Arg-(Arg)-(Arg ).

Reversible trifluoroacetic acid-induced conformational changes in glycophorin as detected by proton nuclear magnetic resonance spectroscopy

High-field (270 MHz) 1H-NMR has been employed to study the solution conformation of glycophorin A, a sialoglycoprotein which spans the human erythrocyte membrane. Glycophorin A is one of the most fully characterized integral membrane proteins known, making it an excellent model for the study of membrane-bound proteins. This protein consists of three distinct domains: a glycosylated extracellular N-terminus, a hydrophobic intramembranous segment, and a polar cytoplasmic C-terminus. These domains contain aromatic residues which serve as convenient 1H-NMR conformational probes. The aromatic region of the NMR spectrum of glycophorin A in 2H 2O shows single, well-resolved His and Tyr resonances. No resonances are observed, however, for the Phe residues which are located in or near the hydrophobic domain. These observations suggest that considerable heterogeneity with respect to segmental motions exists within the protein. This is consistent with circular dichroism data showing the intramembranous segment to be completely helical with the extremities of the protein being predominantly random coils. The helix of the hydrophrobic domain is remarkably resistant to conventional denaturing conditions including variations in pH, and temperature, and treatment with guanidine hydrochloride. However, in trifluoroacetic acid, which strongly solvates peptide backbones, there is extensive reversible unfolding of the helical structure as evidenced by the appearance of Phe resonances. Solvent titration experiments indicate that approximately a 1 : 1 volume ratio of trifluoroacetic acid to 2H 2O is required to initiate unfolding of the helix.

The intramembranous segment of human erythrocyte glycophorin A

The intramembranous segment of glycophorin A has been localized to a 35-amino acid peptide. This has been isolated by a new procedure in which acid-insoluble peptides of a tryptic digest of detergent-purified glycophorin A are fractionated by countercurrent distribution. Amino acid sequence analyses, using both manual and automatic Edman degradation techniques, indicate that this peptide has a unique sequence in contrast to earlier work ( J. P. Segrest, I. Kahane, R. L. Jackson, and V. T. Marchesi, 1973, Biochem. Biophys. Res. Commun., 49, 964–969). Ambiguities at three positions have been resolved, and sequencing errors at two additional positions have been corrected. One segment of this peptide has an uninterrupted stretch of 22 uncharged amino acids, and it is likely that this is the part which spans the lipid bilayer of the membrane. The complete 35-residue peptide has an apparent molecular weight in the 6000–8000 range, when analyzed on sodium dodecyl sulfate gels, suggesting that it forms dimers under these conditions. This result is consistent with our earlier proposal that intact glycophorin A molecules exist as dimers in sodium dodecyl sulfate which are stabilized by noncovalent associations between hydrophobic segments of their polypeptide chains.

The structure of intrinsic membrane proteins.

Intrinsic membrane proteins are embedded in the lipid bilayer so that the polypeptides come in contact with the non-polar region of the bilayer. There are two major types of intrinsic proteins: those with most of their mass outside the cytoplasm (Type I) and those with most of their mass inside the cytoplasm (Type II). In the latter group are the membrane transport systems. The anion exchange system of the human erythrocyte is a dimer of band 3 polypeptides. These polypeptides span the bilary, have most of their mass in the cytoplasm, and are glycosylated. About 20-25% of the polypeptide, however, is in the bilayer. Arguments are presented to support the view that the intramembrane segments of the protein are alpha-helical and that the major protein-protein interactions between the subunits are in the cytoplasmic portion of the protein.