scholarly journals KCNE3 Truncation Mutants Reveal a Bipartite Modulation of KCNQ1 K+ Channels

2004 ◽  
Vol 124 (6) ◽  
pp. 759-771 ◽  
Author(s):  
Steven D. Gage ◽  
William R. Kobertz
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Channel Gating ◽  
Terminal Region ◽  
Type I ◽  
Long Qt ◽  
Transmembrane Peptides ◽  
K Channels ◽  
Voltage Gated ◽  
La Luna

The five KCNE genes encode a family of type I transmembrane peptides that assemble with KCNQ1 and other voltage-gated K+ channels, resulting in potassium conducting complexes with varied channel-gating properties. It has been recently proposed that a triplet of amino acids within the transmembrane domain of KCNE1 and KCNE3 confers modulation specificity to the peptide, since swapping of these three residues essentially converts the recipient KCNE into the donor (Melman, Y.F., A. Domenech, S. de la Luna, and T.V. McDonald. 2001. J. Biol. Chem. 276:6439–6444). However, these results are in stark contrast with earlier KCNE1 deletion studies, which demonstrated that a COOH-terminal region, highly conserved between KCNE1 and KCNE3, was responsible for KCNE1 modulation of KCNQ1 (Tapper, A.R., and A.L. George. 2000 J. Gen. Physiol. 116:379–389.). To ascertain whether KCNE3 peptides behave similarly to KCNE1, we examined a panel of NH2- and COOH-terminal KCNE3 truncation mutants to directly determine the regions required for assembly with and modulation of KCNQ1 channels. Truncations lacking the majority of their NH2 terminus, COOH terminus, or mutants harboring both truncations gave rise to KCNQ1 channel complexes with basal activation, a hallmark of KCNE3 modulation. These results demonstrate that the KCNE3 transmembrane domain is sufficient for assembly with and modulation of KCNQ1 channels and suggests a bipartite model for KCNQ1 modulation by KCNE1 and KCNE3 subunits. In this model, the KCNE3 transmembrane domain is active in modulation and overrides the COOH terminus' contribution, whereas the KCNE1 transmembrane domain is passive and reveals COOH-terminal modulation of KCNQ1 channels. We furthermore test the validity of this model by using the active KCNE3 transmembrane domain to functionally rescue a nonconducting, yet assembly and trafficking competent, long QT mutation located in the conserved COOH-terminal region of KCNE1.

scholarly journals Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

2008 ◽  
Vol 132 (6) ◽  
pp. 681-692 ◽  
Author(s):  
Qinghuan Xiao ◽  
Andrew Prussia ◽  
Kuai Yu ◽  
Yuan-yuan Cui ◽  
H. Criss Hartzell
Keyword(s):  
Amino Acids ◽  
Density Functional ◽  
Transmembrane Domain ◽  
Channel Gating ◽  
Regulatory Domain ◽  
Acidic Amino Acids ◽  
C Terminus ◽  
Ef Hand ◽  
Cl Channels

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl− ion channels that are activated by intracellular Ca2+. At present, the structures and mechanisms responsible for Ca2+ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca2+ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca2+ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca2+ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca2+. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca2+ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca2+-binding loop (312–323) reduced the apparent Ca2+ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca2+-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca2+ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca2+ and suggest a model in which Ca2+ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ∼100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca2+ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca2+.

scholarly journals Determination of Peptide Contact Points in the Human Angiotensin II Type I Receptor (AT1) with Photosensitive Analogs of Angiotensin II

1999 ◽  
Vol 13 (4) ◽  
pp. 578-586 ◽  
Author(s):  
Stéphane A. Laporte ◽  
Antony A. Boucard ◽  
Guy Servant ◽  
Gaétan Guillemette ◽  
Richard Leduc ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Angiotensin Ii ◽  
Transmembrane Domain ◽  
At1 Receptor ◽  
High Yield ◽  
Type I ◽  
Terminal Amino Acid ◽  
Contact Points ◽  
Terminal Amino

Abstract To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-l-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the[ Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166–199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of[ Sar1, Bpa8]AngII, whereas the N-terminal amino acid of[ Bpa1]AngII interacts with the second extracellular loop of the AT1 receptor.

scholarly journals Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 678
Author(s):  
Carter R. Murry ◽  
Irina V. Agarkova ◽  
Jayadri S. Ghosh ◽  
Fiona C. Fitzgerald ◽  
Roger M. Carlson ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Amino Acids ◽  
Dna Sequences ◽  
Transmembrane Domain ◽  
Potassium Ion ◽  
K Channel ◽  
K Channels ◽  
Large Plaque ◽  
Channel Proteins ◽  
Dsdna Viruses

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K+) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K+ channel proteins consisting of 82 amino acids. The KcvATCV-1 protein has similarities to the family of two transmembrane domain K+ channel proteins; it consists of two transmembrane α-helixes with a pore region in the middle, making it an ideal model for studying K+ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from KcvATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K+ channels.

scholarly journals The HOOK region of voltage-gated Ca2+ channel β subunits senses and transmits PIP2 signals to the gate

2017 ◽  
Vol 149 (2) ◽  
pp. 261-276 ◽  
Author(s):  
Cheon-Gyu Park ◽  
Yongsoo Park ◽  
Byung-Chang Suh
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Binding Assay ◽  
Channel Gating ◽  
Inactivation Kinetics ◽  
Β Subunit ◽  
Voltage Gated ◽  
Anionic Phospholipids ◽  
Channel Inhibition ◽  
Important Regulator

The β subunit of voltage-gated Ca2+ (CaV) channels plays an important role in regulating gating of the α1 pore-forming subunit and its regulation by phosphatidylinositol 4,5-bisphosphate (PIP2). Subcellular localization of the CaV β subunit is critical for this effect; N-terminal–dependent membrane targeting of the β subunit slows inactivation and decreases PIP2 sensitivity. Here, we provide evidence that the HOOK region of the β subunit plays an important role in the regulation of CaV biophysics. Based on amino acid composition, we broadly divide the HOOK region into three domains: S (polyserine), A (polyacidic), and B (polybasic). We show that a β subunit containing only its A domain in the HOOK region increases inactivation kinetics and channel inhibition by PIP2 depletion, whereas a β subunit with only a B domain decreases these responses. When both the A and B domains are deleted, or when the entire HOOK region is deleted, the responses are elevated. Using a peptide-to-liposome binding assay and confocal microscopy, we find that the B domain of the HOOK region directly interacts with anionic phospholipids via polybasic and two hydrophobic Phe residues. The β2c-short subunit, which lacks an A domain and contains fewer basic amino acids and no Phe residues in the B domain, neither associates with phospholipids nor affects channel gating dynamically. Together, our data suggest that the flexible HOOK region of the β subunit acts as an important regulator of CaV channel gating via dynamic electrostatic and hydrophobic interaction with the plasma membrane.

scholarly journals In silico and functional characterisation of an ultra-rare CFTR mutation identifies novel lasso motif interactions regulating channel gating

2021 ◽  
Author(s):  
Sharon Wong ◽  
Nikhil Awatade ◽  
Miro Astore ◽  
Katelin Allan ◽  
Michael Carnell ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Transmembrane Domain ◽  
Novel Mutation ◽  
Short Circuit ◽  
Channel Gating ◽  
Short Circuit Current ◽  
Extended Model ◽  
Type I ◽  
Functional Characterisation ◽  
Trafficking Defects

Characterisation of I37R, a novel mutation in the lasso motif of ABC-transporter CFTR, a chloride channel, was conducted by theratyping using CFTR potentiators which increase channel gating activity and correctors which repair protein trafficking defects. I37R-CFTR function was characterised using intestinal current measurements (ICM) in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids and short circuit current measurements (Isc) in organoid-derived monolayers from an individual with I37R/F508del CFTR genotype. We demonstrated that the I37R-CFTR mutation results in a residual function defect amenable to treatment with potentiators and type III, but not to type I, correctors. Molecular dynamics of I37R-CFTR using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavourable strengthening of the interactions between the lasso motif, the regulatory (R) domain and the transmembrane domain two (TMD2). In conclusion, structural and functional characterisation of the I37R-CFTR mutation increases understanding of CFTR channel regulation and provides a potential pathway to access CFTR modulator treatments for individuals with CF caused by ultra-rare CFTR mutations.

scholarly journals KCNE Peptides Differently Affect Voltage Sensor Equilibrium and Equilibration Rates in KCNQ1 K+ Channels

2007 ◽  
Vol 131 (1) ◽  
pp. 59-68 ◽  
Author(s):  
Jessica M. Rocheleau ◽  
William R. Kobertz
Keyword(s):  
Pulse Duration ◽  
Conformational Changes ◽  
Chemical Reactivity ◽  
Voltage Sensor ◽  
Type I ◽  
Transmembrane Peptides ◽  
K Channels ◽  
Voltage Sensors ◽  
State Dependent ◽  
Voltage Dependent

KCNQ1 voltage-gated K+ channels assemble with the family of KCNE type I transmembrane peptides to afford membrane-embedded complexes with diverse channel gating properties. KCNQ1/KCNE1 complexes generate the very slowly activating cardiac IKs current, whereas assembly with KCNE3 produces a constitutively conducting complex involved in K+ recycling in epithelia. To determine whether these two KCNE peptides influence voltage sensing in KCNQ1 channels, we monitored the position of the S4 voltage sensor in KCNQ1/KCNE complexes using cysteine accessibility experiments. A panel of KCNQ1 S4 cysteine mutants was expressed in Xenopus oocytes, treated with the membrane-impermeant cysteine-specific reagent 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and the voltage-dependent accessibility of each mutant was determined. Of these S4 cysteine mutants, three (R228C, G229C, I230C) were modified by MTSET only when KCNQ1 was depolarized. We then employed these state-dependent residues to determine how assembly with KCNE1 and KCNE3 affects KCNQ1 voltage sensor equilibrium and equilibration rates. In the presence of KCNE1, MTSET modification rates for the majority of the cysteine mutants were ∼10-fold slower, as was recently reported to indicate that the kinetics of the KCNQ1 voltage sensor are slowed by KCNE1 (Nakajo, K., and Y. Kubo. 2007 J. Gen. Physiol. 130:269–281). Since MTS modification rates reflect an amalgam of reagent accessibility, chemical reactivity, and protein conformational changes, we varied the depolarization pulse duration to determine whether KCNE1 slows the equilibration rate of the voltage sensors. Using the state-dependent cysteine mutants, we determined that MTSET modification rates were essentially independent of depolarization pulse duration. These results demonstrate that upon depolarization the voltage sensors reach equilibrium quickly in the presence of KCNE1 and the slow gating of the channel complex is not due to slowly moving voltage sensors. In contrast, all cysteine substitutions in the S4 of KCNQ1/KCNE3 complexes were freely accessible to MTSET independent of voltage, which is consistent with KCNE3 shifting the voltage sensor equilibrium to favor the active state at hyperpolarizing potentials. In total, these results suggest that KCNE peptides differently modulate the voltage sensor in KCNQ1 K+ channels.

scholarly journals Identification of the R1 Oncogene and Its Protein Product from the Rhadinovirus of Rhesus Monkeys

1999 ◽  
Vol 73 (6) ◽  
pp. 5123-5131 ◽  
Author(s):  
Blossom Damania ◽  
Mengtao Li ◽  
Joong-Kook Choi ◽  
Louis Alexander ◽  
Jae U. Jung ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Interleukin 2 ◽  
Extracellular Domain ◽  
Open Reading Frame ◽  
Type I ◽  
Reading Frame ◽  
R1 Gene

ABSTRACT Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that is most closely related to the human Kaposi’s sarcoma-associated herpesvirus (KSHV). We have identified a distinct open reading frame at the left end of RRV and designated it R1. The position of the R1 gene is equivalent to that of the saimiri transforming protein (STP) of herpesvirus saimiri (HVS) and of K1 of KSHV, other members of the gamma-2 or rhadinovirus subgroup of herpesviruses. The R1 sequence revealed an open reading frame encoding a product of 423 amino acids that was predicted to contain an extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic tail reflective of a type I membrane-bound protein. The predicted structural motifs of R1, including the presence of immunoreceptor tyrosine-based activation motifs, resembled those in K1 of KSHV but were distinct from those of STP. R1 sequences from four independent isolates from three different macaque species revealed 0.95 to 7.3% divergence over the 423 amino acids. Variation was located predominantly within the predicted extracellular domain. The R1 protein migrated at 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was extensively glycosylated. Tagged R1 protein was localized to the cytoplasmic and plasma membranes of transfected cells. Expression of the R1 gene in Rat-1 fibroblasts induced morphologic changes and focus formation, and injection of R1-expressing cells into nude mice induced the formation of multifocal tumors. A recombinant herpesvirus in which the STP oncogene of HVS was replaced by R1 immortalized T lymphocytes to interleukin-2-independent growth. These results indicate that R1 is an oncogene of RRV.

scholarly journals K+ channel gating: C-type inactivation is enhanced by calcium or lanthanum outside

2014 ◽  
Vol 144 (3) ◽  
pp. 221-230 ◽  
Author(s):  
Clay M. Armstrong ◽  
Toshinori Hoshi
Keyword(s):  
Channel Gating ◽  
Selectivity Filter ◽  
K Channel ◽  
Slow Process ◽  
K Channels ◽  
Inward Current ◽  
Voltage Gated ◽  
Positive Shift ◽  
High Affinity ◽  
Opening And Closing

Many voltage-gated K+ channels exhibit C-type inactivation. This typically slow process has been hypothesized to result from dilation of the outer-most ring of the carbonyls in the selectivity filter, destroying this ring’s ability to bind K+ with high affinity. We report here strong enhancement of C-type inactivation upon extracellular addition of 10–40 mM Ca2+ or 5–50 µM La3+. These multivalent cations mildly increase the rate of C-type inactivation during depolarization and markedly promote inactivation and/or suppress recovery when membrane voltage (Vm) is at resting levels (−80 to −100 mV). At −80 mV with 40 mM Ca2+ and 0 mM K+ externally, ShBΔN channels with the mutation T449A inactivate almost completely within 2 min or less with no pulsing. This behavior is observed only in those mutants that show C-type inactivation on depolarization and is distinct from the effects of Ca2+ and La3+ on activation (opening and closing of the Vm-controlled gate), i.e., slower activation of K+ channels and a positive shift of the mid-voltage of activation. The Ca2+/La3+ effects on C-type inactivation are antagonized by extracellular K+ in the low millimolar range. This, together with the known ability of Ca2+ and La3+ to block inward current through K+ channels at negative voltage, strongly suggests that Ca2+/La3+ acts at the outer mouth of the selectivity filter. We propose that at −80 mV, Ca2+ or La3+ ions compete effectively with K+ at the channel’s outer mouth and prevent K+ from stabilizing the filter’s outer carbonyl ring.

scholarly journals AbeTx1 Is a Novel Sea Anemone Toxin with a Dual Mechanism of Action on Shaker-Type K+ Channels Activation

Marine Drugs ◽  
2018 ◽  
Vol 16 (10) ◽  
pp. 360 ◽  
Author(s):  
Diego B. Orts ◽  
Steve Peigneur ◽  
Laíz Silva-Gonçalves ◽  
Manoel Arcisio-Miranda ◽  
José P. W. Bicudo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cyclic Peptides ◽  
Complex Mixture ◽  
Sea Anemone ◽  
Random Coil ◽  
Cone Snail ◽  
K Channels ◽  
Voltage Gated ◽  
Kv Channels ◽  
Secondary Structure Analysis

Voltage-gated potassium (KV) channels regulate diverse physiological processes and are an important target for developing novel therapeutic approaches. Sea anemone (Cnidaria, Anthozoa) venoms comprise a highly complex mixture of peptide toxins with diverse and selective pharmacology on KV channels. From the nematocysts of the sea anemone Actinia bermudensis, a peptide that we named AbeTx1 was purified and functionally characterized on 12 different subtypes of KV channels (KV1.1–KV1.6; KV2.1; KV3.1; KV4.2; KV4.3; KV11.1; and, Shaker IR), and three voltage-gated sodium channel isoforms (NaV1.2, NaV1.4, and BgNaV). AbeTx1 was selective for Shaker-related K+ channels and is capable of inhibiting K+ currents, not only by blocking the K+ current of KV1.2 subtype, but by altering the energetics of activation of KV1.1 and KV1.6. Moreover, experiments using six synthetic alanine point-mutated analogs further showed that a ring of basic amino acids acts as a multipoint interaction for the binding of the toxin to the channel. The AbeTx1 primary sequence is composed of 17 amino acids with a high proportion of lysines and arginines, including two disulfide bridges (Cys1–Cys4 and Cys2–Cys3), and it is devoid of aromatic or aliphatic amino acids. Secondary structure analysis reveals that AbeTx1 has a highly flexible, random-coil-like conformation, but with a tendency of structuring in the beta sheet. Its overall structure is similar to open-ended cyclic peptides found on the scorpion κ-KTx toxins family, cone snail venoms, and antimicrobial peptides.

Methionine-Specific Staining of Collagen

1982 ◽  
Vol 40 ◽  
pp. 294-295
Author(s):  
E.M. Kuhn ◽  
K.D. Marenus ◽  
M. Beer
Keyword(s):  
Amino Acids ◽  
Collagen Fibers ◽  
Type Iii Collagen ◽  
Type I ◽  
Electron Microscopic ◽  
Good Correspondence ◽  
Specific Staining ◽  
Type Iii ◽  
Different Types ◽  
Sulfur Containing

Fibers composed of different types of collagen cannot be differentiated by conventional electron microscopic stains. We are developing staining procedures aimed at identifying collagen fibers of different types.Pt(Gly-L-Met)Cl binds specifically to sulfur-containing amino acids. Different collagens have methionine (met) residues at somewhat different positions. A good correspondence has been reported between known met positions and Pt(GLM) bands in rat Type I SLS (collagen aggregates in which molecules lie adjacent to each other in exact register). We have confirmed this relationship in Type III collagen SLS (Fig. 1).

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