scholarly journals The Human Heart and Rat Brain IIA Na+ Channels Interact with Different Molecular Regions of the β1 Subunit

2002 ◽  
Vol 120 (6) ◽  
pp. 887-895 ◽  
Author(s):  
Thomas Zimmer ◽  
Klaus Benndorf
Keyword(s):  
Rat Brain ◽  
Human Heart ◽  
Xenopus Oocytes ◽  
Extracellular Domain ◽  
Na Channel ◽  
Na Channels ◽  
Membrane Anchor ◽  
Α Subunit ◽  
Extracellular Region ◽  
Recovery From Inactivation

The α subunit of voltage-gated Na+ channels of brain, skeletal muscle, and cardiomyocytes is functionally modulated by the accessory β1, but not the β2 subunit. In the present study, we used β1/β2 chimeras to identify molecular regions within the β1 subunit that are responsible for both the increase of the current density and the acceleration of recovery from inactivation of the human heart Na+ channel (hH1). The channels were expressed in Xenopus oocytes. As a control, we coexpressed the β1/β2 chimeras with rat brain IIA channels. In agreement with previous studies, the β1 extracellular domain sufficed to modulate IIA channel function. In contrast to this, the extracellular domain of the β1 subunit alone was ineffective to modulate hH1. Instead, the putative membrane anchor plus either the intracellular or the extracellular domain of the β1 subunit was required. An exchange of the β1 membrane anchor by the corresponding β2 subunit region almost completely abolished the effects of the β1 subunit on hH1, suggesting that the β1 membrane anchor plays a crucial role for the modulation of the cardiac Na+ channel isoform. It is concluded that the β1 subunit modulates the cardiac and the neuronal channel isoforms by different molecular interactions: hH1 channels via the membrane anchor plus additional intracellular or extracellular regions, and IIA channels via the extracellular region only.

Block of Human Heart hH1 Sodium Channels by the Enantiomers of Bupivacaine

2000 ◽  
Vol 93 (4) ◽  
pp. 1022-1033 ◽  
Author(s):  
Carla Nau ◽  
Sho-Ya Wang ◽  
Gary R. Strichartz ◽  
Ging Kuo Wang
Keyword(s):  
Human Heart ◽  
Point Mutations ◽  
Cell Voltage ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Amino Acid Residues ◽  
Na Channels ◽  
State Dependent ◽  
Positively Charged

Background S(-)-bupivacaine reportedly exhibits lower cardiotoxicity but similar local anesthetic potency compared with R(+)-bupivacaine. The bupivacaine binding site in human heart (hH1) Na+ channels has not been studied to date. The authors investigated the interaction of bupivacaine enantiomers with hH1 Na+ channels, assessed the contribution of putatively relevant residues to binding, and compared the intrinsic affinities to another isoform, the rat skeletal muscle (mu1) Na+ channel. Methods Human heart and mu1 Na+ channel alpha subunits were transiently expressed in HEK293t cells and investigated during whole cell voltage-clamp conditions. Using site-directed mutagenesis, the authors created point mutations at positions hH1-F1760, hH1-N1765, hH1-Y1767, and hH1-N406 by introducing the positively charged lysine (K) or the negatively charged aspartic acid (D) and studied their influence on state-dependent block by bupivacaine enantiomers. Results Inactivated hH1 Na+ channels displayed a weak stereoselectivity with a stereopotency ratio (+/-) of 1.5. In mutations hH1-F1760K and hH1-N1765K, bupivacaine affinity of inactivated channels was reduced by approximately 20- to 40-fold, in mutation hH1-N406K by approximately sevenfold, and in mutations hH1-Y1767K and hH1-Y1767D by approximately twofold to threefold. Changes in recovery of inactivated mutant channels from block paralleled those of inactivated channel affinity. Inactivated hH1 Na+ channels exhibited a slightly higher intrinsic affinity than mu1 Na+ channels. Conclusions Differences in bupivacaine stereoselectivity and intrinsic affinity between hH1 and mu1 Na+ channels are small and most likely of minor clinical relevance. Amino acid residues in positions hH1-F1760, hH1-N1765, and hH1-N406 may contribute to binding of bupivacaine enantiomers in hH1 Na+ channels, whereas the role of hH1-Y1767 remains unclear.

scholarly journals Arrhythmogenic Mechanism of an LQT-3 Mutation of the Human Heart Na+Channel α-Subunit

Circulation ◽  
2000 ◽  
Vol 102 (5) ◽  
pp. 584-590 ◽  
Author(s):  
X. H. T. Wehrens ◽  
H. Abriel ◽  
C. Cabo ◽  
J. Benhorin ◽  
R. S. Kass
Keyword(s):  
Human Heart ◽  
Na Channel ◽  
Α Subunit

Cloning and functional studies of splice variants of the α-subunit of the amiloride-sensitive Na+ channel

1998 ◽  
Vol 274 (4) ◽  
pp. C1081-C1089 ◽  
Author(s):  
J. Kevin Tucker ◽  
Kaichiro Tamba ◽  
Young-Jae Lee ◽  
Li-Ling Shen ◽  
David G. Warnock ◽  
...  
Keyword(s):  
Amino Acid ◽  
Splice Variants ◽  
Extracellular Domain ◽  
Na Channel ◽  
Wild Type ◽  
Rt Pcr ◽  
Α Subunit ◽  
Functional Studies ◽  
Human Lung Cell Line ◽  
Lung Cell Line

The α-subunit of the amiloride-sensitive epithelial Na+ channel (αENaC) is critical in forming an ion conductive pore in the membrane. We have identified the wild-type and three splice variants of the human αENaC (hαENaC) from the human lung cell line H441, using RT-PCR. These splice variants contain various structures in the extracellular domain, resulting in premature truncation (hαENaCx), 19-amino acid deletion (hαENaC−19), and 22-amino acid insertion (hαENaC+22). Wild-type hαENaC and splice variants were functionally characterized in Xenopus oocytes by coexpression with hENaC β- and γ-subunits. Unlike wild-type hαENaC, undetectable or substantially reduced amiloride-sensitive currents were observed in oocytes expressing these splice variants. Wild-type hαENaC was the most abundantly expressed hαENaC mRNA species in all tissues in which its expression was detected. These findings indicate that the extracellular domain is important to generate structural and functional diversity of hαENaC and that alternative splicing may play a role in regulating hENaC activity.

scholarly journals Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification.

1992 ◽  
Vol 99 (1) ◽  
pp. 1-20 ◽  
Author(s):  
G K Wang ◽  
S Y Wang
Keyword(s):  
Time Course ◽  
Cell Voltage ◽  
Gh3 Cells ◽  
Na Channel ◽  
Na Channels ◽  
H Infinity ◽  
Recovery From Inactivation ◽  
Na Currents ◽  
Steady State Inactivation ◽  
Pharmacological Modification

Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.

Expression of the Human Colonic Na+ Channel α-Subunit in Xenopus Oocytes

1996 ◽  
Vol 90 (s34) ◽  
pp. 6P-7P ◽  
Author(s):  
EH Baker ◽  
RP Boot-Handford ◽  
GI Sandle
Keyword(s):  
Xenopus Oocytes ◽  
Na Channel ◽  
Α Subunit

Relationship between energy expenditure and ion channel density in the turtle and rat brain

1989 ◽  
Vol 257 (6) ◽  
pp. R1354-R1358 ◽  
Author(s):  
R. A. Edwards ◽  
P. L. Lutz ◽  
D. G. Baden
Keyword(s):  
Energy Consumption ◽  
Energy Expenditure ◽  
Ion Channel ◽  
Rat Brain ◽  
Receptor Interaction ◽  
Na Channel ◽  
Apparent Difference ◽  
Na Channels ◽  
Channel Density ◽  
Rat Synaptosomes

Synaptosomes were isolated from turtle and rat brains to determine whether differences in brain ion channel densities accounted for the turtle's ability to survive anoxia compared with the mammal. The Na(+)-channel binding neurotoxin brevetoxin showed high-affinity specific binding in both turtle and rat synaptosomes, suggesting specific ligand-receptor interaction. The maximum binding capacity (Bmax) value for the turtle was only about one-third of that found for the rat synaptosomes, suggesting that the turtle synaptosome has a correspondingly lower Na+ channel density than the rat. This apparent difference in Na+ channel density is not reflected in metabolic rates, since at the same temperature (31 degrees C) the O2 consumption of both the rat and turtle synaptosome was almost identical. The large reductions in energy expenditure seen in synaptosomes incubated in Na(+)-free media and in media containing ouabain (approximately 50% turtle, 80% rat) are probably related to the halting of transmembrane Na(+)-K+ exchange. The greater reduction in the rat may be related to the apparent greater density of Na+ channels in the rat brain. However, compared with the 90% reduction in brain metabolism that occurs when the turtle brain becomes anoxic, the differences in ion channel density and in the costs of ion pumping between the rat and turtle brain are trivial. Closing Na+ channels with tetrodotoxin and increasing Na+ channel activation with veratridine caused substantial decreases and increases in synaptosome energy consumption, respectively. This suggests that the modulation of ion channel conductance has a significant effect on metabolic cost and may be an important mechanism to reduce energy consumption and electrical activity in the anoxic turtle brain, while still maintaining ionic gradients.

scholarly journals High Ca2+ permeability of a peptide-gated DEG/ENaC from Hydra

2012 ◽  
Vol 140 (4) ◽  
pp. 391-402 ◽  
Author(s):  
Stefan Dürrnagel ◽  
Björn H. Falkenburger ◽  
Stefan Gründer
Keyword(s):  
Xenopus Oocytes ◽  
Divalent Cations ◽  
Na Channel ◽  
Low Permeability ◽  
Xenopus Laevis Oocytes ◽  
Na Channels ◽  
Transient Component ◽  
Epithelial Na Channels ◽  
Hydra Magnipapillata ◽  
Secondary Activation

Degenerin/epithelial Na+ channels (DEG/ENaCs) are Na+ channels that are blocked by the diuretic amiloride. In general, they are impermeable for Ca2+ or have a very low permeability for Ca2+. We describe here, however, that a DEG/ENaC from the cnidarian Hydra magnipapillata, the Hydra Na+ channel (HyNaC), is highly permeable for Ca2+ (PCa/PNa = 3.8). HyNaC is directly gated by Hydra neuropeptides, and in Xenopus laevis oocytes expressing HyNaCs, RFamides elicit currents with biphasic kinetics, with a fast transient component and a slower sustained component. Although it was previously reported that the sustained component is unselective for monovalent cations, the selectivity of the transient component had remained unknown. Here, we show that the transient current component arises from secondary activation of the Ca2+-activated Cl− channel (CaCC) of Xenopus oocytes. Inhibiting the activation of the CaCC leads to a simple on–off response of peptide-activated currents with no apparent desensitization. In addition, we identify a conserved ring of negative charges at the outer entrance of the HyNaC pore that is crucial for the high Ca2+ permeability, presumably by attracting divalent cations to the pore. At more positive membrane potentials, the binding of Ca2+ to the ring of negative charges increasingly blocks HyNaC currents. Thus, HyNaC is the first member of the DEG/ENaC gene family with a high Ca2+ permeability.

scholarly journals Augmentation of Recovery from Inactivation by Site-3 Na Channel Toxins

1999 ◽  
Vol 113 (2) ◽  
pp. 333-346 ◽  
Author(s):  
G. Richard Benzinger ◽  
Gayle S. Tonkovich ◽  
Dorothy A. Hanck
Keyword(s):  
Time Course ◽  
Genetic Diseases ◽  
Low Frequency ◽  
Periodic Paralysis ◽  
Na Channel ◽  
Na Channels ◽  
Cell Recovery ◽  
Reverse Transcription Pcr ◽  
Recovery From Inactivation ◽  
Voltage Dependent

Site-3 toxins isolated from several species of scorpion and sea anemone bind to voltage-gated Na channels and prolong the time course of INa by interfering with inactivation with little or no effect on activation, effects that have similarities to those produced by genetic diseases in skeletal muscle (myotonias and periodic paralysis) and heart (long QT syndrome). Some published reports have also reported the presence of a noninactivating persistent current in site-3 toxin-treated cells. We have used the high affinity site-3 toxin Anthopleurin B to study the kinetics of this current and to evaluate kinetic differences between cardiac (in RT4-B8 cells) and neuronal (in N1E-115 cells) Na channels. By reverse transcription–PCR from N1E-115 cell RNA multiple Na channel transcripts were detected; most often isolated were sequences homologous to rBrII, although at low frequency sequences homologous to rPN1 and rBrIII were also detected. Toxin treatment induced a voltage-dependent plateau current in both isoforms for which the relative amplitude (plateau current/peak current) approached a constant value with depolarization, although the magnitude was much greater for neuronal (17%) than cardiac (5%) INa. Cell-attached patch recordings revealed distinct quantitative differences in open times and burst durations between isoforms, but for both isoforms the plateau current comprised discrete bursts separated by quiescent periods, consistent with toxin induction of an increase in the rate of recovery from inactivation rather than a modal failure of inactivation. In accord with this hypothesis, toxin increased the rate of whole-cell recovery at all tested voltages. Moreover, experimental data support a model whereby recovery at negative voltages is augmented through closed states rather than through the open state. We conclude that site-3 toxins produce qualitatively similar effects in cardiac and neuronal channels and discuss implications for channel kinetics.

scholarly journals Action potentials in Xenopus oocytes triggered by blue light

2020 ◽  
Vol 152 (5) ◽  
Author(s):  
Florian Walther ◽  
Dominic Feind ◽  
Christian vom Dahl ◽  
Christoph Emanuel Müller ◽  
Taulant Kukaj ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Blue Light ◽  
Action Potentials ◽  
Xenopus Oocytes ◽  
Na Channel ◽  
Xenopus Laevis Oocytes ◽  
Na Channels ◽  
Excitable Cells ◽  
Voltage Gated

Voltage-gated sodium (Na+) channels are responsible for the fast upstroke of the action potential of excitable cells. The different α subunits of Na+ channels respond to brief membrane depolarizations above a threshold level by undergoing conformational changes that result in the opening of the pore and a subsequent inward flux of Na+. Physiologically, these initial membrane depolarizations are caused by other ion channels that are activated by a variety of stimuli such as mechanical stretch, temperature changes, and various ligands. In the present study, we developed an optogenetic approach to activate Na+ channels and elicit action potentials in Xenopus laevis oocytes. All recordings were performed by the two-microelectrode technique. We first coupled channelrhodopsin-2 (ChR2), a light-sensitive ion channel of the green alga Chlamydomonas reinhardtii, to the auxiliary β1 subunit of voltage-gated Na+ channels. The resulting fusion construct, β1-ChR2, retained the ability to modulate Na+ channel kinetics and generate photosensitive inward currents. Stimulation of Xenopus oocytes coexpressing the skeletal muscle Na+ channel Nav1.4 and β1-ChR2 with 25-ms lasting blue-light pulses resulted in rapid alterations of the membrane potential strongly resembling typical action potentials of excitable cells. Blocking Nav1.4 with tetrodotoxin prevented the fast upstroke and the reversal of the membrane potential. Coexpression of the voltage-gated K+ channel Kv2.1 facilitated action potential repolarization considerably. Light-induced action potentials were also obtained by coexpressing β1-ChR2 with either the neuronal Na+ channel Nav1.2 or the cardiac-specific isoform Nav1.5. Potential applications of this novel optogenetic tool are discussed.

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