Point mutations in alpha bENaC regulate channel gating, ion selectivity, and sensitivity to amiloride

Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.

Download Full-text

Changes in sodium channel gating produced by point mutations in a cytoplasmic linker

Science ◽

10.1126/science.2173138 ◽

1990 ◽

Vol 250 (4981) ◽

pp. 688-691 ◽

Cited By ~ 64

Author(s):

Moorman ◽

G. Kirsch ◽

A. Brown ◽

R. Joho

Keyword(s):

Sodium Channel ◽

Channel Gating ◽

Point Mutations

Download Full-text

Gating mechanisms of a natural anion channelrhodopsin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1513602112 ◽

2015 ◽

Vol 112 (46) ◽

pp. 14236-14241 ◽

Cited By ~ 36

Author(s):

Oleg A. Sineshchekov ◽

Elena G. Govorunova ◽

Hai Li ◽

John L. Spudich

Keyword(s):

Amino Acid ◽

Positive Charge ◽

Ion Selectivity ◽

Channel Gating ◽

Structural Basis ◽

Amino Acid Residues ◽

Gating Mechanisms ◽

Anion Conductance ◽

Guillardia Theta ◽

Sensitive Group

Anion channelrhodopsins (ACRs) are a class of light-gated channels recently identified in cryptophyte algae that provide unprecedented fast and powerful hyperpolarizing tools for optogenetics. Analysis of photocurrents generated byGuillardia thetaACR 1 (GtACR1) and its mutants in response to laser flashes showed thatGtACR1 gating comprises two separate mechanisms with opposite dependencies on the membrane voltage and pH and involving different amino acid residues. The first mechanism, characterized by slow opening and fast closing of the channel, is regulated by Glu-68. Neutralization of this residue (the E68Q mutation) specifically suppressed this first mechanism, but did not eliminate it completely at high pH. Our data indicate the involvement of another, yet-unidentified pH-sensitive group X. Introducing a positive charge at the Glu-68 site (the E68R mutation) inverted the channel gating so that it was open in the dark and closed in the light, without altering its ion selectivity. The second mechanism, characterized by fast opening and slow closing of the channel, was not substantially affected by the E68Q mutation, but was controlled by Cys-102. The C102A mutation reduced the rate of channel closing by the second mechanism by ∼100-fold, whereas it had only a twofold effect on the rate of the first. The results show that anion conductance by ACRs has a fundamentally different structural basis than the relatively well studied conductance by cation channelrhodopsins (CCRs), not attributable to simply a modification of the CCR selectivity filter.

Download Full-text

Optimal transport and colossal ionic mechano-conductance in graphene crown ethers

Science Advances ◽

10.1126/sciadv.aaw5478 ◽

2019 ◽

Vol 5 (7) ◽

pp. eaaw5478 ◽

Cited By ~ 7

Author(s):

Subin Sahu ◽

Justin Elenewski ◽

Christoph Rohmann ◽

Michael Zwolak

Keyword(s):

Ion Transport ◽

Crown Ethers ◽

Optimal Transport ◽

Ion Selectivity ◽

Point Mutations ◽

Diffusion Limit ◽

Macromolecular Systems ◽

Small Pore ◽

Nanofluidic Devices ◽

Pore Domain

Biological ion channels balance electrostatic and dehydration effects to yield large ion selectivity alongside high transport rates. These macromolecular systems are often interrogated through point mutations of their pore domain, limiting the scope of mechanistic studies. In contrast, we demonstrate that graphene crown ether pores afford a simple platform to directly investigate optimal ion transport conditions, i.e., maximum current densities and selectivity. Crown ethers are known for selective ion adsorption. When embedded in graphene, however, transport rates lie below the drift-diffusion limit. We show that small pore strains (1%) give rise to a colossal (100%) change in conductance. This process is electromechanically tunable, with optimal transport in a primarily diffusive regime, tending toward barrierless transport, as opposed to a knock-on mechanism. These observations suggest a novel setup for nanofluidic devices while giving insight into the physical foundation of evolutionarily optimized ion transport in biological pores.

Download Full-text

Detection of Ki-ras gene point mutations in the bile precipitate of patients with various cause of billiary obstruction

Gastroenterology ◽

10.1016/s0016-5085(01)82854-7 ◽

2001 ◽

Vol 120 (5) ◽

pp. A574-A574

Author(s):

C CHEN ◽

S SHIESH ◽

H TSAO ◽

X LIN

Keyword(s):

Point Mutations ◽

Ras Gene

Download Full-text

Fiberoptic DNA sensor array capable of detecting point mutations

Biosensors and Bioelectronics ◽

10.1016/s0956-5663(97)84360-6 ◽

1998 ◽

Vol 13 (2) ◽

pp. iv-v ◽

Cited By ~ 1

Author(s):

B Healey

Keyword(s):

Sensor Array ◽

Point Mutations ◽

Dna Sensor

Download Full-text

A Novel Mutation Glyl672→Arg in Type 2A and a Homozygous Mutation in Type 2B von Willebrand Disease

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650564 ◽

1996 ◽

Vol 76 (02) ◽

pp. 253-257 ◽

Cited By ~ 14

Author(s):

Takeshi Hagiwara ◽

Hiroshi Inaba ◽

Shinichi Yoshida ◽

Keiko Nagaizumi ◽

Morio Arai ◽

...

Keyword(s):

Missense Mutation ◽

Novel Mutation ◽

Point Mutations ◽

Japanese Patients ◽

Von Willebrand Disease ◽

Homozygous Mutation ◽

Missense Mutations ◽

Reaction Products ◽

Type 3 ◽

Von Willebrand

SummaryGenetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.

Download Full-text