Functional reconstitution of the purified brain sodium channel in planar lipid bilayers.

Hartshorne, Robert P.; Keller, Bernhard U.; Talvenheimo, Jane; Catterall, William A.; Montal, Mauricio

doi:10.1073/pnas.82.1.240

Cited by 144 publications

(84 citation statements)

References 30 publications

(27 reference statements)

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“…Recently, methods have been developed to incorporate native sodium channels into artificial planar bilayers by vesicle fusion (15); these channels can be activated by neurotoxins such as batrachotoxin and exhibit single-channel properties in these bilayers comparable to those observed in their native environment (15,16). Single-channel recordings demonstrating voltage-dependent cation conductance have been reported recently for purified sodium channels from eel (17) and from rat brain (18). A limitation of this approach, however, is that only a small number of channels can be studied, and the relationship between the behavior of these channels and the properties of all of the purified channel protein can only be inferred.…”

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confidence: 99%

Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes.

Furman¹,

Tanaka²,

Müeller³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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We have examined the voltage-dependent gating of batrachotoxin-modiflied sodium channels purified from rabbit T-tubular membranes in two ways. First, purified channels were reconstituted into planar bilayers and singlechannel properties were measured. Batrachotoxin-activated channels showed steep voltage-dependent activation with halfmaximal opening probabilities at potentials between -95 and -116 mV. The single-channel conductance (500 mM Na' cis, 200 mM Na' trans) averaged 20 pS and was independent of membrane potential. Channels usually inserted with their extracellular faces on the trans side of the bilayer; addition of tetrodotoxin to the cis side had no effect, whereas addition to the trans side blocked >95% of channel openings at -77 mV.A second approach was used to establish that this voltage dependence was a characteristic of the entire population of purified channels and not just those few channels observed in planar bilayers. Channels reconstituted into egg phosphatidylcholine vesicles were functionally oriented by inclusion of internal saxitoxin; vesicle membrane potentials were then generated by K+ gradients in the presence of valinomycin.After batrachotoxin activation, Vm was altered by shifts of KO'All of the specific 22Na+ influx activated by batrachotoxin and blocked by saxitoxin was found to be voltage sensitive, activating between predicted membrane potentials of -100 and -50 mV. The single-channel properties of the purified Ttubular sodium channel correspond closely to those seen with native sodium channels from rat sarcolemma. The voltagedependent activation of the batrachotoxin-modified reconstituted channel is the same as that seen with native channels in situ or in bilayers after exposure to this toxin. Most importantly, this voltage-dependent gating is a property of all of the purified channels capable of specific pharmacological activation.Voltage-sensitive sodium ion channels play a key role in the generation of action potentials in nerve and skeletal muscle. This ion channel has now been isolated from eel electroplax (1), rat skeletal muscle (2) and brain (3), rabbit skeletal muscle (4), and rat heart (5). In each case the isolated protein contained one large glycoprotein subunit of -260 kDa apparent molecular mass on NaDodSO4/PAGE; the mammalian channels may also contain one or more smaller subunits of -38 kDa (4-9). These proteins have been reconstituted into lipid vesicles and each is able to gate cation fluxes in response to pharmacological activation and block (10-13).A characteristic feature of the sodium channel is its capacity for activation in response to changes in membrane potential. The distinctive voltage-dependent currents recorded from large populations of sodium channels with conventional techniques are reflected at the single-channel level in the voltage-sensitive probability of channel opening events (14). Recently, methods have been developed to incorporate native sodium channels into artificial planar bilayers by vesicle fusion (15); these channels can be a...

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confidence: 99%

Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes.

Furman¹,

Tanaka²,

Müeller³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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confidence: 99%

“…7). Most of these purified preparations have now been reconstituted into artificial membranes and have been shown to possess most of the functional properties of the sodium channel observed in vivo (8)(9)(10)(11)(12).Recently, cDNAs encoding the large glycoprotein from Electrophorus and rat brain have been cloned and sequenced (13,14); further analysis of internal protein sequence homology has shown the presence of four large repeating domains within the 1820-amino acid primary sequence. Subsequent sequence analysis has identified in each of these domains five to seven hydrophobic segments and an additional segment having the unusual property that every third amino acid residue is basic.…”

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confidence: 99%

Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide.

Gordon¹,

Fieles²,

Schotland³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies; noninnervated membrane was not labeled, consistent with the known distribution of sodium channels in this tissue. The membrane topology of the peptide recognized by this antiserum was probed in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% "right-side-out" as determined by [3H]saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. Gold particles identifying the antibody were found almost exclusively associated with the cytoplasmic surface of the innervated membrane. Our data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by our antiserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.The voltage-dependent sodium channel is a transmembrane glycoprotein that regulates the sodium current necessary for generation of action potentials (1). Recent biochemical research has resulted in purification ofthe sodium channel from Electrophorus electricus (2), mammalian skeletal muscle (3, 4), rat brain (5), and chicken heart (6). A finding common to all these reports is that the purified sodium channel contains a glycoprotein subunit of 260-300 kDa, associated in most mammalian preparations with one or two smaller glycoproteins of 33-39 kDa (refs. 3-5; see also ref. 7). Most of these purified preparations have now been reconstituted into artificial membranes and have been shown to possess most of the functional properties of the sodium channel observed in vivo (8)(9)(10)(11)(12).Recently, cDNAs encoding the large glycoprotein from Electrophorus and rat brain have been cloned and sequenced (13,14); further analysis of internal protein sequence homology has shown the presence of four large repeating domains within the 1820-amino acid primary sequence. Subsequent sequence analysis has identified in each of these domains five to seven hydrophobic segments and an additional segment having the unusual property that every third amino acid resi...

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“…Jane Talvenheimo purified and reconstituted sodium channels in Seattle, shipped them to San Diego, and Bob, Bernhard Keller, and Mauricio Montal recorded the functional activity of pure batrachotoxin-activated sodium channels after fusion of reconstituted vesicles with planar bilayers for the first time ( Fig. 2D) (20). To our delight, single sodium channel currents appeared with the conductance and voltage dependence expected for sodium channels activated by batrachotoxin.…”

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confidence: 99%

“…Tetrodotoxin, saxitoxin, and scorpion toxins bind to the ␣ subunits of sodium channels as indicated and were used as molecular tags to identify and purify the sodium channel protein (10,13,14). D, single channel currents conducted by a single purified sodium channel incorporated into a planar bilayer (20).…”

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confidence: 99%

Finding Channels

Catterall¹

2015

Journal of Biological Chemistry

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T his Reflections article tells the story of the early work in my laboratory at the University of Washington that led to discovery of the sodium and calcium channel proteins, followed by a briefer description of the structure and function of these remarkable membrane proteins that has emerged from research in my laboratory and others over many years. I began my scientific career as an undergraduate chemistry major at Brown University, where my senior thesis research with Dr. Joseph Steim introduced me to the mysteries of membrane proteins in [1967][1968]. Little was known about membrane proteins in those days, and the techniques to investigate them were crude by modern standards. Nevertheless, my efforts to solubilize and separate membrane proteins from red blood cells kindled a life-long research interest. As a graduate student in physiological chemistry with Dr. Peter L. Pedersen at Johns Hopkins Medical School in 1968 -1972, my thesis research focused on the F 1 -ATPase, the peripheral membrane component of the ATP synthase of mitochondria. Our work joined the results from other laboratories to establish the complex nine-subunit architecture of that key protein using the newly developed SDS-PAGE method. We also determined some of the substrate binding and functional properties of F 1 -ATPase. A seminar class on neurotransmitters and their receptors with Dr. Solomon Snyder, then an assistant professor of pharmacology at Johns Hopkins, began an interest in neurobiology, which I have continued to pursue. As a postdoctoral fellow with Dr. Marshall Nirenberg at the Laboratory of Biochemical Genetics in the National Heart, Lung, and Blood Institute (NHLBI) at the National Institutes of Health, I moved into neurobiology and molecular pharmacology. Following his Nobel Prize-winning research on the genetic code, Nirenberg had pioneered studies of molecular and cellular neurobiology using cultured neuroblastoma cells as an experimental system. My initial postdoctoral research project was to develop biochemical methods to investigate the voltage-gated sodium channels of neuroblastoma cells. It was a challenging project that led directly to my continuing research interest in the voltage-gated ion channel proteins.Voltage-gated ion channels are responsible for action potential generation in neurons, myocytes, and other excitable cells. They participate in many forms of regulation in other cell types. In excitable cells, action potentials typically are initiated by activation of voltage-gated sodium channels, which conduct sodium rapidly into the cell and depolarize the cell membrane potential. Depolarization activates voltage-gated calcium channels, which conduct calcium into the cell. Calcium entry sustains the depolarization of the cell membrane and generates intracellular calcium transients that initiate many intracellular events, including contraction, secretion, synaptic transmission, regulation of enzymes, and gene expression. Action potentials are terminated by activation of voltage-gated potassium channels, which ...

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Functional reconstitution of the purified brain sodium channel in planar lipid bilayers.

Cited by 144 publications

References 30 publications

Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes.

Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes.

Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide.

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