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

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.

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

Anesthesiology ◽

10.1097/00000542-200010000-00026 ◽

2000 ◽

Vol 93 (4) ◽

pp. 1022-1033 ◽

Cited By ~ 48

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.

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Long-term regulation of renal Na-dependent cotransporters and ENaC: response to altered acid-base intake

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.3.f459 ◽

2000 ◽

Vol 279 (3) ◽

pp. F459-F467 ◽

Cited By ~ 36

Author(s):

Gheun-Ho Kim ◽

Stephen W. Martin ◽

Patricia Fernández-Llama ◽

Shyama Masilamani ◽

Randall K. Packer ◽

...

Keyword(s):

Collecting Duct ◽

Na Channel ◽

Thick Ascending Limb ◽

Acid Base Balance ◽

Acid Base ◽

Opposite Pattern ◽

Α Subunit ◽

Base Balance ◽

Acid Loading

Increased systemic acid intake is associated with an increase in apical Na/H exchange in the renal proximal tubule mediated by the type 3 Na/H exchanger (NHE3). Because NHE3 mediates both proton secretion and Na absorption, increased NHE3 activity could inappropriately perturb Na balance unless there are compensatory changes in Na handling. In this study, we use semiquantitative immunoblotting of rat kidneys to investigate whether acid loading is associated with compensatory decreases in the abundance of renal tubule Na transporters other than NHE3. Long-term (i.e., 7-day) acid loading with NH4Cl produced large decreases in the abundances of the thiazide-sensitive Na-Cl cotransporter (TSC/NCC) of the distal convoluted tubule and both the β- and γ-subunits of the amiloride-sensitive epithelial Na channel (ENaC) of the collecting duct. In addition, the renal cortical abundance of the proximal type 2 Na-dependent phosphate transporter (NaPi-2) was markedly decreased. In contrast, abundances of the bumetanide-sensitive Na-K-2Cl cotransporter of the thick ascending limb and the α-subunit of ENaC were unchanged. A similar profile of changes was seen with short-term (16-h) acid loading. Long-term (7-day) base loading with NaHCO3resulted in the opposite pattern of response with marked increases in the abundances of the β- and γ-subunits of ENaC and NaPi-2. These adaptations may play critical roles in the maintenance in Na balance when changes in acid-base balance occur.

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Comparison of slow inactivation in human heart and rat skeletal muscle Na+channel chimaeras

The Journal of Physiology ◽

10.1111/j.1469-7793.1999.061ad.x ◽

1999 ◽

Vol 515 (1) ◽

pp. 61-73 ◽

Cited By ~ 32

Author(s):

John P. O'Reilly ◽

Sho-Ya Wang ◽

Roland G. Kallen ◽

Ging Kuo Wang

Keyword(s):

Skeletal Muscle ◽

Human Heart ◽

Na Channel ◽

Slow Inactivation ◽

Rat Skeletal Muscle

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On the Interaction between Amiloride and Its Putative α-Subunit Epithelial Na+Channel Binding Site

Journal of Biological Chemistry ◽

10.1074/jbc.m503500200 ◽

2005 ◽

Vol 280 (28) ◽

pp. 26206-26215 ◽

Cited By ~ 26

Author(s):

Ossama B. Kashlan ◽

Shaohu Sheng ◽

Thomas R. Kleyman

Keyword(s):

Binding Site ◽

Na Channel ◽

Α Subunit ◽

Epithelial Na Channel

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Regulation of a cloned epithelial Na+ channel by its β- and γ-subunits

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.6.c1889 ◽

1997 ◽

Vol 273 (6) ◽

pp. C1889-C1899 ◽

Cited By ~ 37

Author(s):

Mouhamed S. Awayda ◽

Albert Tousson ◽

Dale J. Benos

Keyword(s):

Fluorescence Intensity ◽

Expression System ◽

Fold Increase ◽

Na Channel ◽

Β Subunit ◽

Open Probability ◽

Animal Pole ◽

Α Subunit ◽

Amino Acid Peptide ◽

Xenopus Oocyte Expression

Using the Xenopus oocyte expression system, we examined the mechanisms by which the β- and γ-subunits of an epithelial Na+channel (ENaC) regulate α-subunit channel activity and the mechanisms by which β-subunit truncations cause ENaC activation. Expression of α-ENaC alone produced small amiloride-sensitive currents (−43 ± 10 nA, n = 7). These currents increased >30-fold with the coexpression of β- and γ-ENaC to −1,476 ± 254 nA ( n = 20). This increase was accompanied by a 3.1- and 2.7-fold increase of membrane fluorescence intensity in the animal and vegetal poles of the oocyte, respectively, with use of an antibody directed against the α-subunit of ENaC. Truncation of the last 75 amino acids of the β-subunit COOH terminus, as found in the original pedigree of individuals with Liddle’s syndrome, caused a 4.4-fold ( n = 17) increase of the amiloride-sensitive currents compared with wild-type αβγ-ENaC. This was accompanied by a 35% increase of animal pole membrane fluorescence intensity. Injection of a 30-amino acid peptide with sequence identity to the COOH terminus of the human β-ENaC significantly reduced the amiloride-sensitive currents by 40–50%. These observations suggest a tonic inhibitory role on the channel’s open probability ( P o) by the COOH terminus of β-ENaC. We conclude that the changes of current observed with coexpression of the β- and γ-subunits or those observed with β-subunit truncation are likely the result of changes of channel density in combination with large changes of P o.

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Down-regulation of Na channel α-subunit mRNA by protein kinase C e in adrenal chromaffin cells

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)45128-7 ◽

1997 ◽

Vol 73 ◽

pp. 157

Author(s):

Toshihiko Yanagita ◽

Hideyuki Kobayashi ◽

Keizou Masumoto ◽

Ryuichi Yamamoto ◽

Tomoaki Yuhi ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Chromaffin Cells ◽

Na Channel ◽

Adrenal Chromaffin Cells ◽

Down Regulation ◽

Α Subunit ◽

Subunit Mrna ◽

Kinase C ◽

Adrenal Chromaffin

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Regulation of maturation and processing of ENaC subunits in the rat kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00422.2005 ◽

2006 ◽

Vol 291 (3) ◽

pp. F683-F693 ◽

Cited By ~ 102

Author(s):

Zuhal Ergonul ◽

Gustavo Frindt ◽

Lawrence G. Palmer

Keyword(s):

Collecting Duct ◽

Rat Kidney ◽

Na Channel ◽

Cortical Collecting Duct ◽

Α Subunit ◽

Salt Restriction ◽

High K ◽

Electrophysiological Measurements ◽

Immature Form ◽

Epithelial Na Channel

Antibodies directed against subunits of the epithelial Na channel (ENaC) were used together with electrophysiological measurements in the cortical collecting duct to investigate the processing of the proteins in rat kidney with changes in Na or K intake. When animals were maintained on a low-Na diet for 7–9 days, the abundance of two forms of the α-subunit, with apparent masses of 85 and 30 kDa, increased. Salt restriction also increased the abundance of the β-subunit and produced an endoglycosidase H (Endo H)-resistant pool of this subunit. The abundance of the 90-kDa form of the γ-subunit decreased, whereas that of a 70-kDa form increased and this peptide also exhibited Endo H-resistant glycosylation. These changes in α- and γ-subunits were correlated with increases in Na conductance elicited by a 4-h infusion with aldosterone. Changes in all three subunits were correlated with decreases in Na conductance when Na-deprived animals drank saline for 5 h. We conclude that ENaC subunits are mainly in an immature form in salt-replete rats. With Na depletion, the subunits mature in a process that involves proteolytic cleavage and further glycosylation. Similar changes occurred in α- and γ- but not β-subunits when animals were treated with exogenous aldosterone, and in β- and γ- but not α-subunits when animals were fed a high-K diet. Changes in the processing and maturation of the channels occur rapidly enough to be involved in the daily regulation of ENaC activity and Na reabsorption by the kidney.

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Identification of a highly conserved sequence at the N-terminus of the epithelial Na + channel α subunit involved in gating

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s004240051097 ◽

1999 ◽

Vol 438 (5) ◽

pp. 709-715 ◽

Cited By ~ 29

Author(s):

S. Gründer ◽

N. Fowler Jaeger ◽

I. Gautschi ◽

L. Schild ◽

B.C. Rossier

Keyword(s):

Na Channel ◽

Α Subunit ◽

Conserved Sequence ◽

N Terminus ◽

Epithelial Na Channel

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

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.4.c1081 ◽

1998 ◽

Vol 274 (4) ◽

pp. C1081-C1089 ◽

Cited By ~ 15

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.

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