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.

Functional roles of cationic amino acid residues in the sodium-dicarboxylate cotransporter 3 (NaDC-3) from winter flounder

2006 ◽  
Vol 291 (6) ◽  
pp. F1224-F1231 ◽  
Author(s):  
Yohannes Hagos ◽  
Jürgen Steffgen ◽  
Ahsan N. Rizwan ◽  
Denis Langheit ◽  
Ariane Knoll ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transmembrane Helix ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Cationic Amino Acid ◽  
Functional Roles ◽  
Inward Currents ◽  
Induced Currents ◽  
Positively Charged

In the present study, we determined the functional role of 15 positively charged amino acid residues at or within 1 of the predicted 11 transmembrane helixes of the flounder renal sodium-dicarboxylate cotransporter fNaDC-3. Using site-directed mutagenesis, histidine (H), lysine (K), and arginine (R) residues of fNaDC-3 were replaced by alanine (A), isoleucine (I), or leucine (L). Most mutants showed sodium-dependent, lithium-inhibitable [14C]succinate uptake and, in two-electrode voltage-clamp (TEVC) experiments, Km and Δ Imax values comparable to wild-type (WT) fNaDC-3. The replacement of R109 and R110 by alanine and isoleucine (RR109/110AI) prevented the expression of fNaDC-3 at the plasma membrane. When the lysines at positions 232 and 235 were replaced by isoleucine (KK232/235II), the transporter was expressed but showed small transport rates and succinate-induced currents. K114I, located within transmembrane helix 4, showed [14C]succinate uptake similar to WT but relatively small inward currents. When K114 was replaced by arginine, glutamic acid (E), or glutamine (Q), all mutants were expressed at the cell surface. In [14C]succinate uptake and TEVC experiments performed simultaneously on the same oocytes, uptake was similar to or higher than WT, whereas succinate-induced currents were either comparable (K114R) to, or considerably smaller (K114E, K114I, K114Q) than, those evoked by WT. These results suggest that a positively charged residue at position 114 is required for electrogenic sodium-dicarboxylate cotransport.

scholarly journals Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

2021 ◽  
Vol 22 (9) ◽  
pp. 4637
Author(s):  
Daniel Barth ◽  
Andreas Lückhoff ◽  
Frank J. P. Kühn
Keyword(s):  
Amino Acid Residues ◽  
Hek 293 ◽  
Complete Inactivation ◽  
293 Cells ◽  
Activation Mechanisms ◽  
Specific Regulation ◽  
Species Specific ◽  
Inside Out ◽  
Positively Charged

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

Structure and function of voltage-dependent sodium channels: comparison of brain II and cardiac isoforms

1996 ◽  
Vol 76 (3) ◽  
pp. 887-926 ◽  
Author(s):  
H. A. Fozzard ◽  
D. A. Hanck
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Point Mutations ◽  
Amino Acid Sequences ◽  
Na Channel ◽  
Na Channels ◽  
Voltage Dependent ◽  
And Function ◽  
Relationship Of ◽  
The Relationship

Cardiac and nerve Na channels have broadly similar functional properties and amino acid sequences, but they demonstrate specific differences in gating, permeation, ionic block, modulation, and pharmacology. Resolution of three-dimensional structures of Na channels is unlikely in the near future, but a number of amino acid sequences from a variety of species and isoforms are known so that channel differences can be exploited to gain insight into the relationship of structure to function. The combination of molecular biology to create chimeras and channels with point mutations and high-resolution electrophysiological techniques to study function encourage the idea that predictions of structure from function are possible. With the goal of understanding the special properties of the cardiac Na channel, this review examines the structural (sequence) similarities between the cardiac and nerve channels and considers what is known about the relationship of structure to function for voltage-dependent Na channels in general and for the cardiac Na channels in particular.

Epithelial Na channels and short-term renal response to salt deprivation

2002 ◽  
Vol 283 (4) ◽  
pp. F717-F726 ◽  
Author(s):  
Gustavo Frindt ◽  
Tiffany McNair ◽  
Anke Dahlmann ◽  
Emily Jacobs-Palmer ◽  
Lawrence G. Palmer
Keyword(s):  
Distal Tubule ◽  
Treated Group ◽  
Na Channel ◽  
Na Channels ◽  
Nacl Transport ◽  
Drug Concentrations ◽  
Epithelial Na Channels ◽  
Late Evening ◽  
Excretion Rates

To test the role of epithelial Na channels in the day-to-day regulation of renal Na excretion, rats were infused via osmotic minipumps with the Na channel blocker amiloride at rates that achieved drug concentrations of 2–5 μM in the lumen of the distal nephron. Daily Na excretion rates were unchanged, although amiloride-treated animals tended to excrete more Na in the afternoon and less in the late evening than controls. When the rats were given a low-Na diet, Na excretion rates were elevated in the amiloride-treated group within 4 h and remained higher than controls for at least 48 h. Adrenalectomized animals responded similarly to the low-Na diet. In contrast, rats infused with polythiazide at rates designed to inhibit NaCl transport in the distal tubule were able to conserve Na as well as did the controls. Injection of aldosterone (2 μg/100 g body wt) decreased Na excretion in control animals after a 1-h delay. This effect was largely abolished in amiloride-treated rats. On the basis of quantitative analysis of the results, we conclude that activation of amiloride-sensitive channels by mineralocorticoids accounts for 50–80% of the immediate natriuretic response of the kidney to a reduction in Na intake. Furthermore, the channels are necessary to achieve minimal rates of Na excretion during more chronic Na deprivation.

scholarly journals Activation of Ha-ras p21 by substitution, deletion, and insertion mutations.

1985 ◽  
Vol 5 (8) ◽  
pp. 1809-1813 ◽  
Author(s):  
R G Chipperfield ◽  
S S Jones ◽  
K M Lo ◽  
R A Weinberg
Keyword(s):  
Amino Acid ◽  
Point Mutations ◽  
Normal Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Transforming Activity ◽  
Ras P21 ◽  
Ras Oncogenes ◽  
Insertion Mutations

The transforming activity of naturally arising ras oncogenes results from point mutations that affect residue 12 or 61 of the encoded 21-kilodalton protein (p21). By use of site-directed mutagenesis, we showed that deletions and insertions of amino acid residues in the region of residue 12 are also effective in conferring oncogenic activity on p21. Common to these various alterations is the disruption that they create in this domain of the protein, which we propose results in the inactivation of a normal function of the protein.

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.

scholarly journals Isolation from Ochrobactrum anthropi of a Novel Class II 5-Enopyruvylshikimate-3-Phosphate Synthase with High Tolerance to Glyphosate

2010 ◽  
Vol 76 (17) ◽  
pp. 6001-6005 ◽  
Author(s):  
Yong-Sheng Tian ◽  
Ai-Sheng Xiong ◽  
Jing Xu ◽  
Wei Zhao ◽  
Feng Gao ◽  
...  
Keyword(s):  
Genomic Library ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Construction Process ◽  
Ochrobactrum Anthropi ◽  
Gene Encoding ◽  
Phosphate Synthase

ABSTRACT Applying the genomic library construction process and colony screening, a novel aro A gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.

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.

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