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72
The voltage sensor in voltage-dependent ion channels
- Physiol. Rev
, 2000
"... A. The pore and the voltage sensor 556 II. Theoretical Background 559 A. Electric charge movement reflects the operation of the sensor 559 B. Coupling energetics of the sensor and the pore 559 ..."
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A. The pore and the voltage sensor 556 II. Theoretical Background 559 A. Electric charge movement reflects the operation of the sensor 559 B. Coupling energetics of the sensor and the pore 559
Intrinsic disorder and functional proteomics
- Biophys. J
, 2007
"... ABSTRACT The recent advances in the prediction of intrinsically disordered proteins and the use of protein disorder prediction in the fields of molecular biology and bioinformatics are reviewed here, especially with regard to protein function. First, a close look is taken at intrinsically disordered ..."
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Cited by 36 (9 self)
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ABSTRACT The recent advances in the prediction of intrinsically disordered proteins and the use of protein disorder prediction in the fields of molecular biology and bioinformatics are reviewed here, especially with regard to protein function. First, a close look is taken at intrinsically disordered proteins and then at the methods used for their experimental characterization. Next, the major statistical properties of disordered regions are summarized, and prediction models developed thus far are described, including their numerous applications in functional proteomics. The future of the prediction of protein disorder and the future uses of such predictions in functional proteomics comprise the last section of this article.
Mechanism of gating of T-type calcium channels
- J. Gen
, 1990
"... ABSTRACT We have analyzed the gating kinetics of T-type Ca channels in 3T3 fibroblasts. Our results show that channel closing, inactivation, and recovery from inactivation each include a voltage-independent step which becomes rate limiting at extreme potentials. The data require a cyclic model with ..."
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ABSTRACT We have analyzed the gating kinetics of T-type Ca channels in 3T3 fibroblasts. Our results show that channel closing, inactivation, and recovery from inactivation each include a voltage-independent step which becomes rate limiting at extreme potentials. The data require a cyclic model with a minimum of two closed, one open, and two inactivated states. Such a model can produce good fits to our data even if the transitions between closed states are the only voltage-dependent steps in the activating pathway leading from closed to inactivated states. Our analysis suggests that the channel inactivation step, as well as the direct opening and closing transitions, are not intrinsically voltage sensitive. Single-channel recordings are consistent with this scheme. As expected, each channel produces a single burst per opening and then inactivates. Comparison of the kinetics of T-type Ca current in fibroblasts and neuronal cells reveals significant differences which suggest that different subtypes of T-type Ca channels are expressed differentially in a tissue specific manner.
Sodium channel gating in clonal pituitary cells.Journal of General Physiology
, 1989
"... ABSTRACT We have determined the time course of Na channel inactivation in clonal pituitary (GHs) cells by comparing records before and after the enzymatic removal of inactivation. The cells were subjected to whole-cell patch clamp, with papain included in the internal medium. Inactivation was slowly ..."
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ABSTRACT We have determined the time course of Na channel inactivation in clonal pituitary (GHs) cells by comparing records before and after the enzymatic removal of inactivation. The cells were subjected to whole-cell patch clamp, with papain included in the internal medium. Inactivation was slowly removed over the course of 10 min, making it possible to obtain control records before the enzyme acted. Papain caused a large (4-100x) increase in current magnitude for small depolarizations (near-40 mV), and a much smaller increase for large ones (~1.5 • at +40 mV). For technical reasons it was sometimes convenient to study outward IN, recorded with no Na § outside. The instantaneous I-V (IIV) curve in this condition was nonlinear before papain, and more nearly linear afterwards. The gNo-V curve after papain, obtained by dividing the IN,-V curve by the IIV curve, was left-shifted by at least 20 mV and steepened. A spontaneous 5-10 mV left shift occurred in the absence of papaln. The rate of the inactivation step was found to vary only slighdy from-100 mV to +60 mV, based on the following evidence. (a) Before papain, inactivation rate saturated with voltage and was constant from + 20 to + 60 mV. (b) We activated the channels with a brief pulse, and studied the time course of the current on changing the voltage to a second, usually more negative level (Na+ present internally and externally). The time course of inactivation at each voltage was obtained by comparing control traces with those after inactivation was removed. When the 5-10-mV spontaneous shift was taken into account, inactivation rate changed by <10 % from- 100 to +60 mV. The data are considered in terms of existing models of the Na channel.
Surviva l of K* permeability and gating currents in squid axons perfused with K+-free mediaJ. Gen. Physiol
, 1980
"... K + currents were recorded in squid axons internally perfused with impermeant electrolyte. Total absence of permeant ions inside and out leads to an irreversible loss of potassium conductance with a time constant of ~ 11 min at 8 ~ Potassium channels can be protected against this effect by external ..."
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Cited by 22 (2 self)
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K + currents were recorded in squid axons internally perfused with impermeant electrolyte. Total absence of permeant ions inside and out leads to an irreversible loss of potassium conductance with a time constant of ~ 11 min at 8 ~ Potassium channels can be protected against this effect by external K +, Cs +, NH4 +, and Rb + at concentrations of 100-440 mM. These experiments suggest that a K + channel is normally occupied by one or more small cations, and becomes nonfunctional when these cations are removed. A large charge movement said to be related to K + channel gating in frog skeletal muscle is absent in squid giant axons. However, deliberate destruction of K + conductance by removal of permeant cations is accompanied by measurable loss in asymmetric charge movement. This missing charge component is large enough to contain a contribution from K + gating charge movements of more than five elementary charges per channel.
Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation
, 2002
"... abstract The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluoresce ..."
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abstract The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation. key words: conformational changes • fluorescence • gating currents • sodium channel
Developmental changes in Na + conductances in rat neocortical neurons: appearance of a slowly inactivating component
- Journal of
, 1988
"... in rat neocortical neurons from the sensorimotor area. Neurons were obtained by acute dissociation from animals at developmental stages from embryonic day 16 (E16) to postnatal day 50 (P.50) to quantify any developmental changes in the kinetic properties of the Na+ conductance. 2. Neurons were divid ..."
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Cited by 15 (0 self)
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in rat neocortical neurons from the sensorimotor area. Neurons were obtained by acute dissociation from animals at developmental stages from embryonic day 16 (E16) to postnatal day 50 (P.50) to quantify any developmental changes in the kinetic properties of the Na+ conductance. 2. Neurons were divided into two classes, based on morphology, to determine whether there are any cell-type specific differences in Na+ conductances that contribute to the different action potential morphologies seen in current-clamp recordings in vitro. 3. Upon isolation, neurons were voltage clamped using the whole-cell variation of the
Interactions of amino terminal domains of Shaker K channels with a pore blocking site studied with synthetic peptides
- J. Gen. Physiol
, 1993
"... ABSTRACT Synthetic peptides of the five alternative NH2-terminal sequences of Shaker when applied to the cytoplasmic side of ShB channels that have an NH2-terminal deletion (ShBA6-46) block the channel with potencies correlated with the rate of inactivation in the corresponding variant. These peptid ..."
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Cited by 15 (1 self)
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ABSTRACT Synthetic peptides of the five alternative NH2-terminal sequences of Shaker when applied to the cytoplasmic side of ShB channels that have an NH2-terminal deletion (ShBA6-46) block the channel with potencies correlated with the rate of inactivation in the corresponding variant. These peptides share no sequence similarity and yet three out of the five have apparent dissociation constants between 2 and 15 p.M, suggesting that the specificity requirements for binding are low. To identify the primary structural determinants required for effective block of ShBA6-46, we examined the effects of substitutions made to the 20 residue ShB peptide on association and dissociation rates. Nonpolar residues within the peptide appear to be important in stabilizing the binding through hydrophobic interactions. Substitutions to leucine-7 showed there was a clear correlation between hydrophobicity and the dissociation rate constant (koff) with little effect on the association rate constant (kon). Substituting charged residues for hydrophobic residues within the region 4-8 disrupted binding. Within the COOH-terminal half of the peptide, substitutions that increased the net positive charge increased kon with
Inactivation of potassium current in squid axon by a variety of quaternary ammonium ions
- J. Gen. Physiol
, 1981
"... A B $ T R A C T The characteristics of potassium channel block by a diverse group of quaternary ammonium (QA) ions was examined in squid axons. Altering the size and nature of the head and/or tail groups of the QA ions applied internally produced only quantitative differences in the potassium curren ..."
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Cited by 12 (0 self)
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A B $ T R A C T The characteristics of potassium channel block by a diverse group of quaternary ammonium (QA) ions was examined in squid axons. Altering the size and nature of the head and/or tail groups of the QA ions applied internally produced only quantitative differences in the potassium current block. Although their entry rate is diminished, compounds with head groups as large as 11 • 12,~, are capable of occluding the channel, whereas the smallest QA ions, with head groups approximately 5 • 6,~, are not potent blockers. When one or three terminal hydrogens of the head group were replaced by hydroxyl moieties, the compound's blocking ability was diminished, suggesting that QA binding is not improved by hydrogen bonding at these positions. QA ions bound to their site within the potassium channel with 1:1 stoichiometry, and the site is perhaps 20 % or more of the distance through the membrane electric field. Raising external potassium concentration did not alter the steady-state or kinetic features of the QA block of outward potassium currents; however, increasing temperature or adding Ba 2+ internally increased the rate of decay of the QA-blocked currents. From the structure-function analysis of the QA ions, projections concerning both the architecture of the potassium channel's inner mouth and the significance of various chemical constituents of the ions were made. The potassium channel may now be pictured as having a wider mouth (up to 11 • 12,~) extending to the QA binding site and then narrowing quickly to the region of channel selectivity. Important alterations that improve the blocking ability of the compounds include: (a) lengthening the alkyl hydrocarbon tail group (up to 10 carbons), (b) lengthening a second hydrocarbon chain of the head group (e.g., decyldimethylphenylammonium bromide [Cl0DMth]), and (c) adding a carbonyl moiety to the tail (e.g., ambutonium).
Immobilizing the Moving Parts of Voltage-gated Ion Channels
"... abstract Voltage-gated ion channels have at least two classes of moving parts, voltage sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To explore the coupling between voltage sensors and gates, we have syste ..."
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abstract Voltage-gated ion channels have at least two classes of moving parts, voltage sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To explore the coupling between voltage sensors and gates, we have systematically immobilized each using a bifunctional photoactivatable cross-linker, benzophenone-4-carboxamidocysteine methanethiosulfonate, that can be tethered to cysteines introduced into the channel protein by mutagenesis. To validate the method, we first tested it on the inactivation gate of the sodium channel. The benzophenone-labeled inactivation gate of the sodium channel can be trapped selectively either in an open or closed state by ultraviolet irradiation at either a hyperpolarized or depolarized voltage, respectively. To verify that ultraviolet light can immobilize S4 segments, we examined its relative effects on ionic and gating currents in Shaker potassium channels, labeled at residue 359 at the extracellular end of the S4 segment. As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments. Photocross-linking occurs preferably at hyperpolarized voltages after labeling residue 359, suggesting that depolarization moves the benzophenone adduct out of a restricted environment. Immobilization of the S4 segment of the second domain of sodium channels prevents channels from opening. By contrast, photocross-linking the S4 segment of the fourth domain
