Papers by Edward Moczydlowski
The Journal of General Physiology, Apr 26, 2004
Kluwer Academic Publishers-Plenum Publishers eBooks, Dec 9, 2005
Potassium channels are biomolecular devices that mediate the genesis and regulation of membrane v... more Potassium channels are biomolecular devices that mediate the genesis and regulation of membrane voltage, a physiochemical process that is essential to many forms of life. The key to understanding K-channel function lies in understanding the evolution of channel proteins. K-channels exist in nature as a multitude of related modular constructs with unique functional specialties. Analysis of the structural relationships among diverse
Biophysical Journal, Dec 1, 1996
Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly di... more Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains 1, 11, 111, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the ,u1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain Ill of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(lIl) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.
Biophysical Journal, Mar 1, 1991
Mammalian heart Na+ channels exhibit-100-fold higher affinity for block by external Zn2+ than oth... more Mammalian heart Na+ channels exhibit-100-fold higher affinity for block by external Zn2+ than other Na+ channel subtypes. With batrachotoxin-modified Na+ channels from dog or calf heart, micromolar concentrations of external Zn2+ result in a flickering block to a substate level with a conductance of 12% of the open channel at-50 mV. We examined the hypothesis that, in this blocking mode, Zn2+ binds to a subsite of the saxitoxin (STX) binding site of heart Na+ channels by single-channel analysis of the interaction between Zn2+ and STX and also by chemical modification experiments on single heart Na+ channels incorporated into planar lipid bilayers in the presence of batrachotoxin. We found that external Zn2+ relieved block by STX in a strictly competitive fashion. Kinetic analysis of this phenomenon was consistent with a scheme involving direct binding competition between Zn2+ and STX at a single site with intrinsic equilibrium dissociation constants of 30 nM for STX and 30 ,uM for Zn2+. Because high-affinity Zn2+-binding sites often include sulfhydryl groups as coordinating ligands of this metal ion, we tested the effect of a sulfhydryl-specific alkylating reagent, iodoacetamide (IM), on Zn2+ and STX block. For six calf heart Na+ channels, we observed that exposure to 5 mM IM completely abolished Zn2+ block and concomitantly modified STX binding with at least 20-fold reduction in affinity. These results lead us to propose a model in which Zn2+ binds to a subsite within or near the STX binding site of heart Na+ channels. This site is also presumed to contain one or more cysteine sulfhydryl groups.
OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information), 2013
low (55 nM) and [InsP 3 ] was rapidly dropped from 10 mM to 0 using perfusion solution exchanges,... more low (55 nM) and [InsP 3 ] was rapidly dropped from 10 mM to 0 using perfusion solution exchanges, the InsP 3 R channels had a high propensity to transiently enter a more active state before they became inactivated by the removal of InsP 3. This behavior was more prominent, with the channel P o transiently increasing to~0.6, when [Ca 2þ ] i was increased to optimal level (2.1 mM) instead of remaining constant at 55 nM. No transient increase in P o was observed in similar [InsP 3 ] drops with [Ca 2þ ] i kept constant at 2.1 mM. This suggests that sub-saturating [InsP 3 ] can induce type 1 InsP 3 R channels to enter the high-P o mode with higher probability than saturating [InsP 3 ].
Channels, May 19, 2013
Abbreviations: hERG (Kv11.1), human ether-ago go related channel; Kv, voltage-gated K + channel; ... more Abbreviations: hERG (Kv11.1), human ether-ago go related channel; Kv, voltage-gated K + channel; LA, local anesthetic; Nav, voltage-gated Na + channel; TBA + , tetrabutylammonium; TEA + , tetraethylammonium ©2013 Landes Bioscience. Do not distribute.
The Journal of General Physiology, 1996
In this study, we investigated the mechanism underlying the production of inwardly rectifying sub... more In this study, we investigated the mechanism underlying the production of inwardly rectifying subconductance states induced in large conductance CaZ+-activated K § channels (maxi K(Ca) channels) by the small, homologous proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin-I (DTX). Low-resolution bilayer recordings of BPTI-induced substates display excess noise that is well described by a [3-distribution characteristic of a filtered, two-state process. High-resolution patch recordings of maxi K(Ca) channels from vascular smooth muscle cells confirm that the BPTI-induced substate is actually comprised of rapid, voltage-dependent transitions between the open state and a nearly closed state. Patch recordings of DTX-induced substates also exhibit excess noise consistent with a similar two-state fluctuation process that occurs at rates faster than those measured for the BPTI-induced substate. The results indicate that these examples of ligand-induced substates originate by a fluctuating barrier mechanism that is similar to one class of models proposed by Dani,J. A., andJ. A. Fox (1991. J. Theor. Biol. 153:401-423) to explain subconductance behavior of ion channels. To assess the general impact of such rapid fluctuations on the practical measurement of unitary currents by amplitude histograms, we simulated single-channel records for a linear, three-state scheme of C(closed)-O(open)-S(substate). This simulation defines a range of transition rates relative to filter frequency where rapid fluctuations can lead to serious underestimation of actual unitary current levels. On the basis of these experiments and simulations, we conclude that fluctuating barrier processes and open channel noise may play an important physiological role in the modulation of ion permeation.
Biophysical Journal, Mar 1, 1994
Mammalian heart sodium channels inserted into planar bilayers exhibit a distinctive subconductanc... more Mammalian heart sodium channels inserted into planar bilayers exhibit a distinctive subconductance state when single batrachotoxin-modified channels are exposed to external Zn2+. The current-voltage behavior of the open state and the Zn2+-induced substate was characterized in the presence of symmetrical Na+ ranging from 2 to 3000 mM. The unitary conductance of the open state follows a biphasic dependence on [Na+] that can be accounted for by a 3-barrier-2-site model of Na+ permeation that includes double occupancy and Na+-Na+ repulsion. The unitary conductance of the Zn2+ substate follows a monophasic dependence on [Na+] that can be explained by a similar 3-barrier-2-site model with low affinity for Na+ and single occupancy due to repulsive interaction with a Zn2+ ion bound near the external entrance to the pore. The apparent association rate of Zn2+ derived from dwell-time analysis of flickering events is strongly reduced as [Na+] is raised from 50 to 500 mM. The apparent dissociation rate of Zn2+ is also enhanced as [Na+] is increased. While not excluding surface charge effects, such behavior is consistent with two types of ion-ion interactions: 1) A competitive binding interaction between Zn2+ and Na+ due to mutual competition for high affinity sites in close proximity. 2) A noncompetitive, destabilizing interaction resulting from simultaneous occupancy by Zn2+ and Na+. The repulsive influence of Zn2+ on Na+ binding in the cardiac Na+ channel is similar to that which has been proposed to occur between Ca2+ and Na+ in structurally related calcium channels. Based on recent mutagenesis data, a schematic model of functionally important residues in the external cation binding sites of calcium channels and cardiac sodium channels is proposed. In this model, the Zn2+-induced subconductance state results from Zn2+ binding to a site in the external vestibule that is close to the entrance of the pore but does not occlude it.
Journal of Biological Chemistry, Aug 1, 1991
Plasma from the bullfrog, Rana catesbeiana, contains a soluble component of unknown function that... more Plasma from the bullfrog, Rana catesbeiana, contains a soluble component of unknown function that specifically binds the neurotoxin, [3H]saxitoxin, with a Kd of-0.2 nM. Saxiphilin, the protein responsible for this activity, was purified-440-fold from bullfrog plasma by column chromatography on heparin-Sepharose followed by chromatofocusing. The purified saxiphilin preparation exhibits a binding capacity of 9.6 nmol/mg protein and a K d of 0.32 nM for [3H]saxitoxin. Analysis of the preparation by sodium dodecyl sulfatepolyacrylamide gel electrophoresis shows a predominant band migrating with an apparent M , of-89,000 which is similar to the expected size of saxiphilin previously estimated by nondenaturing size exclusion chromatography. Amino-terminal sequencing of the-89-kDa protein and sequencing of four different tryptic peptide fragments revealed that each of the partial saxiphilin sequences can be aligned by homology with members of the transferrin protein family with sequence identity as high as 69%. The available sequence corresponding to conserved residues that comprise part of the two Fe3+ binding sites in lactotransferrin show several substitutions in saxiphilin, suggesting that saxiphilin is not an Fe3'-binding protein. Saxiphilin appears to be a monomeric-89-kDa protein that is evolutionarily related to transferrin but which binds saxitoxin instead of Fe3+.
Journal of Biological Chemistry, Dec 1, 1977
The Journal of General Physiology, Dec 1, 1997
Organic Cation Permeation in Na ϩ Channel Mutants which amino acid residues are likely to form th... more Organic Cation Permeation in Na ϩ Channel Mutants which amino acid residues are likely to form the pore region for several types of channels. This has motivated attempts to determine the relationship between amino acid side chains thought to form the lining or selectivity region of channel pores, and the molecular cutoff behavior with respect to organic ions (Cohen et al., 1992; Wang and Imoto, 1992; Goulding et al., 1993). In this paper, we address a specific question along these lines for the voltage-gated Na ϩ channel of rat skeletal muscle: do key residues that are known to determine ionic selectivity among Na ϩ , K ϩ , and Ca 2 ϩ also control the size-selective permeation of organic cations? Voltage-gated Na ϩ and Ca 2 ϩ channels are homologous proteins that differ in their ionic selectivity. Native Na ϩ channels are at least 10-fold more permeable to Na ϩ than K ϩ and are virtually impermeable to Ca 2 ϩ (Hille, 1972; Campbell, 1976; Pappone, 1980). Ca 2 ϩ channels are highly selective for group IIA divalent cations (Ca 2 ϩ , Sr 2 ϩ , Ba 2 ϩ), but also exhibit nonselective currents of Na ϩ or K ϩ when the concentration of Ca 2 ϩ is quite low (Ͻ 1 M) (Kuo and Hess, 1993; Almers and McCleskey, 1984; Almers et al., 1984; Hess and Tsien, 1984). Site-directed mutagenesis experiments have revealed that the structural basis for this ion discrimination is primarily specified by a conserved motif of four amino acid residues, one in each of the four internally homologous domains that comprise the ␣ subunit of pseudo-tetrameric Na ϩ and Ca 2 ϩ channels (Heine
Journal of Biological Chemistry, Mar 1, 1981
To whom reprint requests should be addressed. the structure and mechanism of the (Na,K) pump are ... more To whom reprint requests should be addressed. the structure and mechanism of the (Na,K) pump are discussed. (Na,K)-ATPase' exhibits complex kinetics of activation with respect to ATP. In the presence of Na' and K', double reciprocal plots of ATPase velocity uersus ATP concentration are convex upward (1-6). By extrapolating linear regions, two apparent K,,, values for ATP and two apparent V,,, values for
Journal of Biological Chemistry, Mar 1, 1981
The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adeno... more The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate (TNP-ATP) binds specifically with enhanced fluorescence to the ATP site of purified eel electroplax sodium-potassium adenosine triphosphatase, (Na,K)-ATPase. A single homogeneous high affinity TNP-ATP binding site with a KD of 0.04 to 0.09 microM at 3 degrees C and 0.2 to 0.7 microM at 21 degrees-25 degrees C was observed in the absence of ligands when binding was measured by fluorescence titration or with [3H]TNP-ATP. ATP and other nucleotides competed with TNP-ATP for binding with KD values similar to those previously determined for binding to the ATP site. Binding stoichiometries determined from Scatchard plot intercepts gave one TNP-ATP site/175,000 g of protein (range: 1.64 X 10(5) to 1.92 X 10(5) when (Na,K)-ATPase protein was determined by quantitative amino acid analysis. The ratio of [3H]ouabain sites to TNP-ATP sites was 0.91. These results are inconsistent with "half-of-sites" binding and suggest that there is one ATP and one ouabain site/alpha beta protomer. (Na,K)-ATPase maintained a high affinity for TNP-ATP regardless of the ligands present. K+ increased the KD for TNP-ATP about 5-fold and Na+ reversed the effect of K+. The effects of Na+, K+, and mg2+ on ATP binding at 3 degrees C were studied fluorimetrically by displacement of TNP-ATP by ATP. The results are consistent with competition between ATP and TNP-ATP for binding at a single site regardless of the metallic ions present. The derived KD values for ATP were : no ligands, 1 microM; 20 mM NaCl, 3-4 microM; 20 mM KCl, 15-19 microM; 20 mM Kcl + 4 mM MgCl2, 70-120 microM. These results suggests that a single ATP site exhibits a high or low affinity for ATP depending on the ligands present, so that high and low affinity ATP sites observed kinetically are interconvertible and do not co-exist independently. We propose that during turnover the affinity for ATP changes more than 100-fold owing to the conformational changes associated with ion binding, translocation, and release.
Springer eBooks, 1986
Biochemical progress toward the mechanism of voltage-dependent Na+ channels has enjoyed the benef... more Biochemical progress toward the mechanism of voltage-dependent Na+ channels has enjoyed the benefit of an extensive molecular pharmacology that includes natural specific toxins as well as synthetic chemical probes of this channel. The recently introduced method of studying chemically activated Na+ channels in planar lipid bilayers with the use of batrachotoxin (Krueger et al., 1983) makes it possible to examine functional manifestations of Na+-channel pharmacology at the level of individual channel macromolecules. In this chapter, we summarize the results of our investigations of the blocking of batrachotoxin-activated Na+ channels from rat skeletal muscle by three classes of pharmacological agents: specific guanidinium toxins, synthetic organic cations, and local anesthetics.
Chemistry & Biology, Nov 1, 1998
The determination of the crystal structure of a K+-selective channel protein from Streptomyces hd... more The determination of the crystal structure of a K+-selective channel protein from Streptomyces hdans reveals how the rapid movement of K+ across membranes is catalyzed by a large family of pore-forming proteins. Many features of the structure mirror hypotheses, predictions and models of K+ channels developed over the past four decades of functional analysis.
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Essential Ion Channel Methods , 2010
Publisher Summary A planar bilayer is an artificial membrane formed across a small hole, ∼50 μm o... more Publisher Summary A planar bilayer is an artificial membrane formed across a small hole, ∼50 μm or larger in diameter. The hole on which the membrane is formed is usually placed in a thin plastic partition separating two aqueous compartments, but artificial bilayers may also be formed on a glass micropipette tip. Insertion or incorporation of a channel-forming molecule into such a membrane provides a simple experimental system for electrical recording of channel-mediated currents. Planar bilayer recording of ion channels is practiced for a number of reasons. It is a technique that yields incredibly rich mechanistic information on a relatively low budget while offering kaleidoscopic displays of single-channel fluctuations that some workers find delightful, even soothing.. This chapter highlights technical aspects of selected examples where the bilayer approach has been widely applied to functional analysis of major classes of ion channel proteins. Identification of techniques and preparations that have been found to be most generally applicable may facilitate extension of the bilayer approach to other classes of channel proteins that have not yet been thoroughly domesticated.
Ion channel proteins regulate complex patterns of cellular electrical activity and ionic signalin... more Ion channel proteins regulate complex patterns of cellular electrical activity and ionic signaling. Certain K + channels play an important role in immunological biodefense mechanisms of adaptive and innate immunity. Most ion channel proteins are oligomeric complexes with the conductive pore located at the central subunit interface. The long-term activity of many K + channel proteins is dependent on the concentration of extracellular K + ; however, the mechanism is unclear. Thus, this project focused on mechanisms underlying structural stability of tetrameric K + channels. Using KcsA of Streptomyces lividans as a model K + channel of known structure, the molecular basis of tetramer stability was investigated by: 1. Bioinformatic analysis of the tetramer interface. 2. Effect of two local anesthetics (lidocaine, tetracaine) on tetramer stability. 3. Molecular simulation of drug docking to the ion conduction pore. The results provide new insights regarding the structural stability of K + channels and its possible role in cell physiology.
Biochemistry, Mar 4, 1980
... Edward G. Moczydlowski and PA George Fortes ... Most studies of cardiac glycoside interaction... more ... Edward G. Moczydlowski and PA George Fortes ... Most studies of cardiac glycoside interaction with Na,K-ATPase have been done by measuring binding of radioactive glycosides (Erdmann & Schoner, 1973a,b; Wallic & Schwartz, 1974; Hansen & Skou, 1973; Choi & Akera, 1977 ...
Springer eBooks, 1986
Until recent years, analysis of ion channel mechanisms belonged to the specialty of electrophysio... more Until recent years, analysis of ion channel mechanisms belonged to the specialty of electrophysiology. Prerequisites for this work included a background in electronics, microelectrode techniques, and voltage clamping. In this voltage-clamp era, the complete repertoire of biochemical techniques had been difficult to apply to channel proteins because there were no routine assays for purification and functional studies. At present, advances in many fields have salvaged ion channels for biochemistry, as evidenced by the diversity of approaches in this volume. Liposome, planar bilayer, and patch recording studies take their inspiration from test-tube tactics that are familiar to many workers in the biochemical and molecular fields. The classical electrophysiologist now hastens to learn the subtleties of channel cloning, while the molecular biologist pauses to reviews the conclusions of noise analysis.
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Papers by Edward Moczydlowski