Molecular design of oligomeric channel proteins.
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Amino Acid Sequence↗
Biomedical subjects
M Montal
Publications and source records attributed to M Montal.
Expression of a genomic clone encoding a brain potassium channel in mammalian cells using lipofection.
A genomic clone encoding a mouse brain K+ channel (MBK1) was isolated, characterized and expressed in COS cells using the lipofection technique. Transfected COS cells expressed voltage-dependent K+ currents that activated within 20 ms at 0 mV and showed less than 10% inactivation during 250 ms depolarizing pulses at 60 mV. Expressed K+ currents were reversibly blocked by 4-aminopyridine and tetraethylammonium, and were moderately sensitive to dendrotoxin, but insensitive to charybdotoxin. Thus MBK1, expressed transiently in a mammalian cell line, exhibits features characteristic of non-inactivating K+ channels with a conspicuous insensitivity to charybdotoxin. Lipofection is, therefore, a valuable strategy for expression of channel proteins in mammalian cells.
Animals↗
Synthetic peptides and proteins as models for pore-forming structure of channel proteins.
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Amino Acid Sequence↗
Mapping of a human brain voltage-gated calcium channel to human chromosome 12p13-pter.
Degenerate DNA oligomers coding for highly conserved regions of the voltage-gated calcium channel were synthesized for the polymerase chain reaction (PCR) using DNA from a human brain cDNA library as template. PCR amplified a 640-bp DNA fragment from the human brain cDNA library. Sequencing revealed that this fragment encodes part of a protein highly homologous to a subtype of the dihydropyridine-sensitive calcium channel cloned from rabbit heart and rat brain. Southern analysis of panels of somatic cell hybrids mapped the 640-bp fragment, CACNL1A1, to human chromosome 12p13-pter.
Amino Acid Sequence↗
Agonist-independent activation of acetylcholine receptor channels by protein kinase A phosphorylation.
Protein phosphorylation is a ubiquitous and one of the most effective means of regulating protein activity. Receptor phosphorylation is a key event in signal transduction. The question, therefore, that arises is whether this modulatory mechanism might produce functional changes in a membrane receptor in the absence of its naturally occurring ligand. To examine this issue, single-channel properties of purified acetylcholine receptors (AChRs) from Torpedo californica reconstituted in lipid bilayers were studied in the absence of ACh in both unphosphorylated preparations and after in vitro phosphorylation by a purified catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A). Notably, the spontaneous open-channel probability of phosphorylated AChRs is significantly higher than that of unphosphorylated AChRs. Channel activation by protein kinase A is correlated with AChR phosphorylation and is abolished by alpha-bungarotoxin. Analysis of probability distributions of the open dwell times indicates that, similar to unphosphorylated AChR has two distinct open states, short- and long-lived. The frequency of occurrence of the long openings over the short and the magnitude of both time constants increase after phosphorylation, as they do with agonist concentration. Thus, phosphorylation of AChR gamma and delta subunits activates AChR channel opening in the absence of ligand binding. This result is compatible with the notion that protein phosphorylation may effectively act as an intracellular ligand with the phosphorylation sites envisioned as cytoplasmic ligand binding sites.
Acetylcholine↗
A molecular blueprint for the pore-forming structure of voltage-gated calcium channels.
A protein that imitates the sequence of a highly conserved segment predicted to line the pore of dihydropyridine-sensitive L-type calcium channels was designed and synthesized. Single-channel conductance properties were studied in planar lipid bilayers. The synthetic protein emulates the ionic conductance, ionic selectivity, and pharmacological properties of the authentic calcium channel, including the stereospecific action of agonist and antagonist enantiomers of the dihydropyridine BayK 8644. The identified sequence is identical in L-type calcium channels from skeletal muscle and isoforms of cardiac muscle, brain, and aorta. It is plausible that this structural motif represents the molecular blueprint for the pore-forming structure of voltage-gated calcium channels.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Orientations of amphipathic helical peptides in membrane bilayers determined by solid-state NMR spectroscopy.
Solid-state NMR spectroscopy was used to determine the orientations of two amphipathic helical peptides associated with lipid bilayers. A single spectral parameter provides sufficient orientational information for these peptides, which are known, from other methods, to be helical. The orientations of the peptides were determined using the 15N chemical shift observed for specifically labeled peptide sites. Magainin, an antibiotic peptide from frog skin, was found to lie in the plane of the bilayer. M2 delta, a helical segment of the nicotinic acetylcholine receptor, was found to span the membrane, perpendicular to the plane of the bilayer. These findings have important implications for the mechanisms of biological functions of these peptides.
Amino Acid Sequence↗
Bundles of amphipathic transmembrane alpha-helices as a structural motif for ion-conducting channel proteins: studies on sodium channels and acetylcholine receptors.
Channel proteins are transmembrane symmetric (or pseudosymmetric) oligomers organized around a central ionic pore. We present here a molecular model of the pore forming structures of two channel proteins with different primary structures and oligomeric size: the voltage-sensitive sodium channel and the nicotinic cholinergic receptor. We report low-energy arrangements of alpha-helical bundles calculated by semiempiricial potential energy functions and optimization routines and further refined using molecular dynamics. The ion-conducting pore is considered to be a symmetric or pseudosymmetric homooligomer of 3-5 amphipathic alpha-helices arranged such that the polar residues line a central hydrophilic pathway and the apolar residues face the hydrophobic bilayer interior. The channel lining exposes either charged (Asp, Glu, Arg, Lys) or polar-neutral (Ser, Thr) residues. A bundle of four parallel helices constrained to C4 symmetry, the helix axis aligned with the symmetry axis, and the helices constrained to idealized dihedral angles, produces a structure with a pore of the size inferred for the sodium channel protein (area approximately 16 A2). Similarly, a pentameric array optimized with constraints to maintain C5 symmetry and backbone torsions characteristic of alpha-helices adopts a structure that appears well suited to form the lining of the nicotinic cholinergic receptor (pore area approximately 46 A2). Thus, bundles of amphipathic alpha-helices satisfy the structural, energetic, and dynamic requirements to be the molecular structural motif underlying the function of ionic channels.
Amino Acid Sequence↗
Tetanus toxin channel in phosphatidylserine planar bilayers: conductance states and pH dependence.
Tetanus toxin (TeTx) forms ionic channel in phosphatidylserine bilayers. TeTx channels exhibit different modes of channel bursting activity, from a closed state to well defined open states of different amplitudes. At positive applied voltages, TeTx channels flicker continuously between a closed state and the various distinct open states. Furthermore, fast transitions into subconductance states are discernible within the bursts of channel activity. Elementary conductance steps submultiple of the open states were not identified in single channel records owing to rapid transitions between different states. However, statistical analysis shows that conductances cluster with amplitudes multiple of an elementary value: e.g. 25-30 pS at neutral pH. Single channel current amplitudes decrease with the pH of the bulk electrolyte solution. Conductance decrements can be accounted for by the relative decrease of permeant cation concentration at the membrane-water interface, by a relative enrichment of protons that block the channel or by the stabilization of a conformational state of the channel protein.
Hydrogen-Ion Concentration↗
Two-dimensional probability density analysis of single channel currents from reconstituted acetylcholine receptors and sodium channels.
Two-dimensional probability density analysis of single channel current recordings was applied to two purified channel proteins reconstituted in planar lipid bilayers: Torpedo acetylcholine receptors and voltage-sensitive sodium channels from rat brain. The information contained in the dynamic history of the gating process, i.e., the time sequence of opening and closing events was extracted from two-dimensional distributions of transitions between identifiable states. This approach allows one to identify kinetic models consistent with the observables. Gating of acetylcholine receptors expresses "memory" of the transition history: the receptor has two channel open (O) states; the residence time in each of them strongly depends on both the preceding open time and the intervening closed interval. Correspondingly, the residence time in the closed (C) states depends on both the preceding open time and the preceding closed time. This result confirms the scheme that considers, at least, two transition pathways between the open and closed states and extends the details of the model in that it defines that the short-lived open state is primarily entered from long-lived closed states while the long-lived open state is accessed mainly through short-lived closed states. Since ligand binding to the acetylcholine-binding sites is a reaction with channel closed states, we infer that the longest closed state (approximately 19 ms) is unliganded, the intermediate closed state (approximately 2 ms) is singly liganded and makes transitions to the short open state (approximately 0.5 ms) and the shortest closed state (approximately 0.4 ms) is doubly liganded and isomerizes to long open states (approximately 5 ms). This is the simplest interpretation consistent with available data. In contrast, sodium channels modified with batrachotoxin to eliminate inactivation show no correlation in the sequence of channel opening and closing events, i.e., have no memory of the transition history. This result is, therefore, consistent with any kinetic scheme that considers a single transition pathway between open and closed states, and confirms the C-C-O model previously inferred from one-dimensional distribution analysis. The strategy described should be of general validity in the analysis of single channel events from channel proteins in both natural and reconstituted membranes.
Animals↗
Voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671.
A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay. The rates of early activation and subsequent inactivation accelerate and approach a maximum in response to test potentials, V, of greater depolarization. The magnitude of peak sodium current increased from negligible values below V = -50 mV and reached a maximum at V = -3.6 mV +/- 2.7 mV (mean +/- S.E.M., n = 12). Sodium currents reversed at V = + 70 mV, near the predicted Nernst equilibrium potential for a Na+ selective channel. The peak sodium conductance, gpeak increased with depolarizing voltages to a maximum at V = approximately 0 mV, exhibiting half-activation voltage at V approximately equal to -36.8 mV and an e-fold change in gpeak/9.5 mV. The Hodgkin-Huxley inactivation parameter h infinity indicates that at V = -73.6 mV half of the sodium currents were inactivated. Single channel current recordings demonstrated the occurrence of discrete events: the latency for first opening was shorter as the depolarizing pulse became more positive. The single-channel current amplitude was ohmic with a slope conductance, gamma = 17.13 pS +/- 0.66 pS. Sodium channel currents were reversibly blocked by tetrodotoxin (TTX).(ABSTRACT TRUNCATED AT 250 WORDS)
Cells, Cultured↗
Synporins--synthetic proteins that emulate the pore structure of biological ionic channels.
A class of proteins that mimic the fundamental pore structure of authentic ionic channels has been designed, synthesized, and characterized. The design is based on our earlier result that a 23-mer peptide with the sequence of the M2 segment of the Torpedo californica acetylcholine receptor delta subunit--Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-Ala-Gln-Ala-Val-Phe -Leu- Leu-Leu-Thr-Ser-Gln-Arg--forms cation-selective channels in lipid bilayers, presumably by self-assembly of conductive oligomers. Accordingly, a tethered parallel tetramer was synthesized with four M2 delta peptides attached to a carrier template--a 9-amino acid backbone with four attachment sites. As expected, the complete 101-residue protein does form channels in lipid bilayers reproducing several features that are characteristic of authentic acetylcholine receptor channels, such as single-channel conductance, cation selectivity, transitions between closed and open states in the millisecond time range, and sensitivity to local anesthetic channel blockers. An analogue protein, in which the serine residue in position 8 is replaced with alanine in each of the four M2 delta 23-mer peptides ([Ala8]M2 delta), also forms channels that, however, exhibit lower single-channel conductance. By contrast, a similar tethered tetramer with M1 delta peptides does not form channels, in accord with expectations. The general validity of this strategy to other channel sequences and oligomer numbers is anticipated. Thus, synporins--a term coined to identify this class of synthetic pore proteins--enrich our armamentarium directed toward the elucidation of structure-function relationships.
Amino Acid Sequence↗
Molecular anatomy and molecular design of channel proteins.
A central goal in membrane biology is to understand how channel proteins work in terms of their underlying protein structures. Ionic channels are symmetric (or pseudosymmetric) transmembrane protein assemblies organized around a central aqueous pore. The two key functional elements are the ionic channel, the actual polar pathway that permits the selective passage of 10(8) ions per second across the apolar core of the membrane lipid bilayer, and the sensor, the structure that detects the stimulus and couples it to the opening or closing (gating) of the channel. The current excitement in membrane protein science emerges from structural information that is providing clues about the molecular determinants of function: molecular cloning and sequencing has led to the elucidation of the primary structures of several superfamilies of voltage-gated and ligand-gated channels; channel proteins have been purified and reconstituted in lipid bilayers with full retention of function; the properties of many channel proteins have been characterized at the single-channel level; cDNA or RNA transcripts have been expressed in oocytes as functional proteins; specific peptide sequences predicted by molecular modeling to form the channel lining have been synthesized by solid-phase methods and proved to be channel formers in lipid bilayers. These advances are beginning to delineate general principles about the molecular design of this class of proteins that are essential for cellular excitability and signal transduction.
Amino Acid Sequence↗
Channel protein engineering. An approach to the identification of molecular determinants of function in voltage-gated and ligand-regulated channel proteins.
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Amino Acid Sequence↗
The M2 delta transmembrane domain of the nicotinic cholinergic receptor forms ion channels in human erythrocyte membranes.
A synthetic peptide with the sequence of the M2 delta segment of the nicotinic acetylcholine receptor from Torpedo californica forms pores in human erythrocyte membranes as determined by hemoglobin and potassium release. This peptide forms a permeability pathway with an apparent cross-sectional diameter of 7-9 A. The M2 delta pore is oligomeric and a pentamer is the species that accounts for the properties of the permeation path. Peptides that mimic other identifiable segments of the Torpedo acetylcholine receptor, M1 delta and MIR, do not form channels in erythrocytes under the same conditions.
Amino Acid Sequence↗
Rhodopsin-G-protein interactions monitored by resonance energy transfer.
Resonance energy transfer measurements were implemented to monitor the specific interactions between G-protein and rhodopsin in phospholipid vesicles reconstituted with the purified proteins. Fluorescently labeled G-protein was extracted from bleached rod outer segments (ROS) reacted with several sulfhydryl reagents: N-(1-pyrenyl)maleimide (P), monobromobimane (B), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (C), and N-(4-anilino-1-naphthyl)maleimide (A). Limited labeling of ROS, resulting in the modification of less than a single -SH residue per G-protein molecule and less than 0.2 residue per rhodopsin, did not impair the specific in situ interactions between rhodopsin and G-protein. This was demonstrated by preservation of their light-activated tight association and Gpp(NH)p binding and their fast dissociation with excess GTP. The distribution of fluorescent label among the three subunits of G-protein revealed a highly reactive -SH group in the gamma subunit accessible to labeling when G-protein was bound specifically to bleached rhodopsin. Recombination of purified fluorescent derivatives of G-protein with purified rhodopsin reconstituted in lipid vesicles restored the light-activated Gpp(NH)p binding to a level comparable to that measured with unlabeled G-protein. Similar observations were obtained with ROS depleted of peripheral proteins. Likewise, modification of up to two -SH groups per rhodopsin molecule with the fluorescent reagents did not affect the functional recombination of G-protein with rhodopsin in reconstituted lipid vesicles or in depleted ROS. Interactions between rhodopsin and G-protein were monitored by resonance energy transfer measurements, with the following fluorescent conjugates as donor/acceptor couples: P-rhodopsin/C-G-protein, P-rhodopsin/B-G-protein, and P-G-protein/C-rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671.
The human neuromedulloblastoma cell line TE671 is shown by single-channel recordings to express nicotinic acetylcholine receptors (AChRs) that are blocked by alpha-bungarotoxin (alpha Bgt). These AChRs do not react with antisera to the alpha Bgt-binding protein of brain or with monoclonal antibodies (mAbs) to brain nicotinic AChRs that do not bind alpha Bgt. TE671 AChRs do react with autoantibodies to muscle AChRs from myasthenia gravis patients and with mAbs to muscle AChRs, including mAbs specific for extrajunctional AChRs. AChRs. AChRs purified from TE671 cells are composed of 4 kinds of subunits corresponding to those of muscle AChR. Sequences of cDNAs for the ACh-binding alpha subunit and the delta subunit of this AChR further identify it as muscle AChR. Expression of TE671 AChR can be up-regulated by nicotine and dexamethasone, and down-regulated by forskolin.
Amino Acid Sequence↗
Characterization of the channel properties of tetanus toxin in planar lipid bilayers.
A detailed characterization of the properties of the channel formed by tetanus toxin in planar lipid bilayers is presented. Channel formation proceeds at neutral pH. However, an acidic pH is required to detect the presence of channels in the membrane rapidly and effectively. Acid pH markedly lowers the single-channel conductance, for phosphatidylserine at 0.5 M KCl gamma = 89 pS at pH 7.0 while at pH 4.8, gamma = 30 pS. The toxin channel is cation selective without significant selectivity between potassium and sodium (gamma [K+]/gamma [Na+] greater than or equal to 1.35). In all the lipids studied gamma is larger at positive than at negative voltages. The toxin channel is voltage dependent both at neutral and acidic pH: for phosphatidylserine membranes, the probability of the channel being open is much greater at positive than at negative voltage. In different phospholipids the channel exhibits different voltage dependence. In phosphatidylserine membranes the channel is inactivated at negative voltages, whereas in diphytanoylphosphatidylcholine membranes channels are more active at negative voltages than at positive. The presence of acidic phospholipids in the bilayers increases both the single-channel conductance as well as the probability of the channel being open at positive voltage. A subconductance state is readily identifiable in the single-channel recordings. Accordingly, single-channel conductance histograms are best fitted with a sum of 3 Gaussian distributions corresponding to the closed state, the open subconductance state and the full open state. Channel activity occurs in bursts of openings separated by long closings. Probability density analysis of the open dwell times of the toxin channel indicate the existence of a single open state with a lifetime greater than or equal to 1 ms in all lipids studied. Analysis of intra-bursts closing lifetimes reveals the existence of two components; the slow component is of the order of 1 ms, the fast one is less than or equal to 0.5 ms. The channel activity induced by tetanus toxin in lipid bilayers suggests a mechanism for its neurotoxicity: a voltage dependent, cation selective channel inserted in the postsynaptic membrane would lead to continuous depolarization and, therefore, persistent activation of the postsynaptic cell.
Electric Conductivity↗