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M H Akabas

Publications and source records attributed to M H Akabas.

30 records · Page 2Linked to original sources
PubMed1994

Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator forms a chloride channel that is regulated by phosphorylation and intracellular ATP levels. The structure of the channel-forming domains is undetermined. To identify the residues lining this channel we substituted cysteine, one at a time, for 9 consecutive residues (91-99) in the M1 membrane-spanning segment. The cysteine substitution mutants were expressed in Xenopus oocytes. We determined the accessibility of the engineered cysteine to charged, sulfhydryl-specific methanethiosulfonate reagents added extracellularly. We assume that, among residues in membrane-spanning segments, only those lining the channel will be accessible to react with these hydrophilic reagents and that such a reaction would irreversibly alter conduction through the channel. Only the cysteines substituted for Gly-91, Lys-95, and Gln-98 were accessible to the reagents. We conclude that these residues are in the channel lining. The periodicity of these residues is consistent with an alpha-helical secondary structure.

Amino Acid Sequence↗
PubMed1994

Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit.

Each residue in and flanking the M2 membrane-spanning segment of the alpha subunit, from Glu-241 to Glu-262, was mutated to cysteine, and the mutant subunits were expressed together with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Cysteines substituted for Glu-262, Leu-258, Val-255, Ser-252, Leu-251, Leu-250, Ser-248, Leu-245, Thr-244, and Glu-241 reacted with the positively charged, hydrophilic, sulfhydryl-specific reagent methanethiosulfonate ethylammonium (MTSEA), added extracellularly. These 10 residues, therefore, are exposed in the channel lumen. The pattern of exposure is compatible with an alpha helix, interrupted by an extended structure from Leu-250 to Ser-252. Acetylcholine caused subtle changes in the accessibilities of some of the engineered cysteines. Since all 10 residues are accessible to MTSEA in the closed state of the channel, the channel gate is at least as cytoplasmic as Glu-241, the most cytoplasmic of the residues tested.

Acetylcholine↗
PubMed1993

Amino acids lining the channel of the gamma-aminobutyric acid type A receptor identified by cysteine substitution.

The binding of gamma-aminobutyric acid (GABA) to gamma-aminobutyric acid type A (GABAA) receptors triggers the opening of an anion-selective channel. To identify amino acid residues that line the channel, we combined cysteine mutagenesis and covalent chemical modification. We mutated, one at a time, four consecutive residues (268-271) in the M2 membrane-spanning segment of the rat GABAA receptor alpha 1 subunit to cysteine and expressed the mutant alpha 1 subunits, together with either the beta 1 subunit or the beta 1 and gamma 2 subunits, in Xenopus oocytes. We probed the susceptibility of the cysteine substitution mutants to covalent modification by charged, sulfhydryl reagents added extracellularly. We assumed that among the residues in membrane-spanning segments, only those lining the channel would be susceptible to modification by polar reagents and that such modification would irreversibly alter conduction. We infer that the residues Thr-268 and Ile-271 are exposed in the channel in both the open and closed states but that Leu-269 and Ser-270 are not exposed. The susceptibility of Thr-268 and Ile-271 in the closed state implies that the gate must be closer to the cytoplasmic end of the channel than Thr-268.

Amino Acid Sequence↗
PubMed1992

Acetylcholine receptor channel structure probed in cysteine-substitution mutants.

In order to understand the structural bases of ion conduction, ion selectivity, and gating in the nicotinic acetylcholine receptor, mutagenesis and covalent modification were combined to identify the amino acid residues that line the channel. The side chains of alternate residues--Ser248, Leu250, Ser252, and Thr254--in M2, a membrane-spanning segment of the alpha subunit, are exposed in the closed channel. Thus alpha 248-254 probably forms a beta strand, and the gate is closer to the cytoplasmic end of the channel than any of these residues. On channel opening, Leu251 is also exposed. These results lead to a revised view of the closed and open channel structures.

Acetylcholine↗
PubMed1990

Mechanisms of chemosensory transduction in taste cells.

The application of new techniques to the study of taste cells has revealed much about both the basic physiology of these cells and also about the mechanisms of taste transduction. The taste cells are electrically excitable cells with a variety of voltage-dependent ion currents. These ionic currents have an important role in the transduction of salt taste in mammals and frogs. In mudpuppies different ion channels are involved in the transduction of acidic-sour stimuli. The role of ion currents in the transduction of sweet taste is less clear. Some proposed mechanisms suggest an important role for ion currents and others suggest that the transduction process may be a biochemical event involving cell surface receptors and intracellular second messengers, possibly cAMP. The transduction of bitter taste seems to be a biochemical event involving cell surface receptors and intracellular second messengers in the inositol trisphosphate pathway. Thus, one cannot talk about "the mechanism" of taste transduction. Different taste modalities are transduced by different mechanisms. A corollary to this is that taste cells are not a homogeneous population of cells. In order to provide animals with the ability to discriminate between different taste modalities the taste cells consist of distinct subpopulations of cells based on their primary taste modality. The primary taste modality in a given cell is determined by the receptors and transduction mechanism(s) expressed in that cell. Evidence suggests that modality-specific receptors are expressed in a segregated manner in distinct subpopulations of taste cells. Secondary responses observed in gustatory axons may arise due to a lack of absolute specificity in the transduction processes and nonspecific effects of low pH and high ionic strength and osmolarity on the taste cells. An interesting area for future work will be to elucidate the mechanism(s) by which basal cells become committed to a given taste modality and how the gustatory neurons influence this process of differentiation. The involvement of the gustatory neurons is critical as they must synapse with taste cells of the correct taste modality to preserve the integrity of the information transferred to the CNS. This process of synaptogenesis is presumably mediated by the expression of taste-modality-specific, cell surface antigens on the basolateral domain of a taste cell and receptors on the appropriate neurons, but much work will be necessary to elucidate this process.(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride↗
PubMed1989

Purification and reconstitution of chloride channels from kidney and trachea.

Chloride channels mediate absorption and secretion of fluid in epithelia, and the regulation of these channels is now known to be defective in cystic fibrosis. Indanyl-oxyacetic acid 94 (IAA-94) is a high-affinity ligand for the chloride channel, and an affinity resin based on that structure was developed. Solubilized proteins from kidney and trachea membranes were applied to the affinity matrix, and four proteins with apparent molecular masses of 97, 64, 40, and 27 kilodaltons were eluted from the column by excess IAA-94. A potential-dependent 36Cl- uptake was observed after reconstituting these proteins into liposomes. Three types of chloride channels with single-channel conductances of 26, 100, and 400 picosiemens were observed after fusion of these liposomes with planar lipid bilayers. Similar types of chloride channels have been observed in epithelia.

Animals↗
PubMed1989

Fusion of phospholipid vesicles with a planar membrane depends on the membrane permeability of the solute used to create the osmotic pressure.

Phospholipid vesicles fuse with a planar membrane when they are osmotically swollen. Channels in the vesicle membrane are required for swelling to occur when the vesicle-containing compartment is made hyperosmotic by adding a solute (termed an osmoticant). We have studied fusion using two different channels, porin, a highly permeable channel, and nystatin, a much less permeable channel. We report that an osmoticant's ability to support fusion (defined as the magnitude of osmotic gradient necessary to obtain sustained fusion) depends on both its permeability through lipid bilayer as well as its permeability through the channel by which it enters the vesicle interior. With porin as the channel, formamide requires an osmotic gradient about ten times that required with urea, which is approximately 1/40th as permeant as formamide through bare lipid membrane. When nystatin is the channel, however, fusion rates sustained by osmotic gradients of formamide are within a factor of two of those obtained with urea. Vesicles containing a porin-impermeant solute can be induced to swell and fuse with a planar membrane when the impermeant bathing the vesicles is replaced by an isosmotic quantity of a porin-permeant solute. With this method of swelling, formamide is as effective as urea in obtaining fusion. In addition, we report that binding of vesicles to the planar membrane does not make the contact region more permeable to the osmoticant than is bare lipid bilayer. In the companion paper, we quantitatively account for the observation that the ability of a solute to promote fusion depends on its permeability properties and the method of swelling. We show that the intravesicular pressure developed drives fusion.

Bacterial Outer Membrane Proteins↗
PubMed1988

A bitter substance induces a rise in intracellular calcium in a subpopulation of rat taste cells.

The sense of taste permits animals to discriminate between foods that are safe and those that are toxic. Because most poisonous plant alkaloids are intensely bitter, bitter taste warns animals of potentially hazardous foods. To investigate the mechanism of bitter taste transduction, a preparation of dissociated rat taste cells was developed that can be studied with techniques designed for single-cell measurements. Denatonium, a very bitter substance, caused a rise in the intracellular calcium concentration due to release from internal stores in a small subpopulation of taste cells. Thus, the transduction of bitter taste may occur via a receptor-second messenger mechanism leading to neurotransmitter release and may not involve depolarization-mediated calcium entry.

Animals↗
PubMed1984

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes.

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.

Hydrocarbons↗
PubMed1984

Separation of the osmotically driven fusion event from vesicle-planar membrane attachment in a model system for exocytosis.

We demonstrate that there are two experimentally distinguishable steps in the fusion of phospholipid vesicles with planar bilayer membranes. In the first step, the vesicles form a stable, tightly bound pre-fusion state with the planar membrane; divalent cations (Ca++) are required for the formation of this state if the vesicular and/or planar membrane contain negatively charged lipids. In the second step, the actual fusion of vesicular and planar membranes occurs. The driving force for this step is the osmotic swelling of vesicles attached (in the pre-fusion state) to the planar membrane. We suggest that osmotic swelling of vesicles may also be crucial for biological fusion and exocytosis.

Calcium↗
PubMed1982

Osmotic swelling of phospholipid vesicles causes them to fuse with a planar phospholipid bilayer membrane.

Fusion of phospholipid vesicles with planar bilayer membranes occurs if the vesicles that contact the planar membrane swell osmotically after the replacement in their medium of an impermeant solute by a permeant one. This finding directly demonstrates that osmotic swelling is a driving force for vesicle-planar membrane fusion. The method used to induce vesicle swelling and fusion may have relevance for biological systems.

Calcium↗