scholarly journals Modular Design of Cys-loop Ligand-gated Ion Channels: Functional 5-HT3 and GABA ρ1 Receptors Lacking the Large Cytoplasmic M3M4 Loop

2008 ◽  
Vol 131 (2) ◽  
pp. 137-146 ◽  
Author(s):  
Michaela Jansen ◽  
Moez Bali ◽  
Myles H. Akabas
Keyword(s):  
Ion Channels ◽  
Acetylcholine Receptors ◽  
Modular Design ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Channel Assembly ◽  
And Function ◽  
Gated Ion Channels

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the α-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT3A) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT3A-glvM3M4 receptors were comparable to 5-HT3A wild type. The channels remained cation-selective but the 5-HT3A-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT3 and acetylcholine receptors, significantly attenuated 5-HT–induced currents with wild-type 5-HT3A but not 5-HT3A-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-ρ1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.

scholarly journals Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the “Pre-M1” Linker

2007 ◽  
Vol 130 (6) ◽  
pp. 559-568 ◽  
Author(s):  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptors ◽  
State Energy ◽  
Transmembrane Domain ◽  
Salt Bridge ◽  
Side Chain ◽  
Coupling Energy ◽  
And Function ◽  
Loop 2 ◽  
Α Subunits ◽  
Α1 Subunit

Charged residues in the β10–M1 linker region (“pre-M1”) are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational “wave.” We examined the effects of mutations to all five residues in pre-M1 (positions M207–P211) plus E45 in loop 2 in the mouse α1-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (Keq), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in Keq (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Φ values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Φ values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only −0.33 kcal/mol (for both α subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.

scholarly journals Functional regions of the mouse interleukin-10 receptor cytoplasmic domain.

1995 ◽  
Vol 15 (9) ◽  
pp. 5043-5053 ◽  
Author(s):  
A S Ho ◽  
S H Wei ◽  
A L Mui ◽  
A Miyajima ◽  
K W Moore
Keyword(s):  
Transmembrane Domain ◽  
Cytoplasmic Domain ◽  
Interleukin 10 ◽  
Proliferation Assay ◽  
Wild Type ◽  
Cell Surface Antigens ◽  
Functional Regions ◽  
Heat Stable ◽  
C Terminus ◽  
Heat Stable Antigen

The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested.

Single-channel recording of ligand-gated ion channels

2007 ◽  
Vol 2 (11) ◽  
pp. 2826-2841 ◽  
Author(s):  
Martin Mortensen ◽  
Trevor G Smart
Keyword(s):  
Ion Channels ◽  
Single Channel ◽  
Single Channel Recording ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

scholarly journals Mutants of the membrane-binding region of Semliki Forest virus E2 protein. II. Topology and membrane binding.

1986 ◽  
Vol 102 (3) ◽  
pp. 902-910 ◽  
Author(s):  
D F Cutler ◽  
P Melancon ◽  
H Garoff
Keyword(s):  
Cell Membranes ◽  
Semliki Forest Virus ◽  
Transmembrane Domain ◽  
Membrane Binding ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Hydrophobic Domain ◽  
E2 Protein ◽  
Amino Terminal ◽  
Charge Cluster

The p62/E2 protein of Semliki Forest virus (SFV) is a typical transmembrane glycoprotein, with an amino-terminal lumenal domain, a transmembrane (hydrophobic) domain, and a carboxy-terminal cytoplasmic domain (or tail). Our hypothesis has been that the membrane-binding polypeptide region (membrane anchor) of this protein consists of both the transmembrane domain and the adjacent positively charged peptide, Arg-Ser-Lys, which is part of the cytoplasmic domain. We have investigated three anchor mutants of the p62 protein with respect to both their disposition and their stability in cell membranes. The construction of the three mutants has been described (Cutler, D.F., and H. Garoff, J. Cell Biol., 102:889-901). They are as follows: A1, changing the basic charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Glu(-1); A2, replacing an Ala in the middle of the hydrophobic stretch with a Glu; A3, changing the charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Met(0). All three mutants retain the transmembrane configuration of the wild-type p62. In a cell homogenate they have a cytoplasmic domain that is accessible to protease. In living cells an anti-peptide antibody specific for the cytoplasmic tail of p62 reacts with the tails of both wild-type and mutant p62s following its introduction into the cytoplasm. All three mutant proteins have Triton X-114 binding properties similar to the wild-type p62. However, when the membranes of cells expressing the three mutants or the wild-type p62 protein are washed with sodium carbonate, pH 11.5, three to four times as much mutant protein as wild-type p62 is released from the membranes. Thus the stability in cell membranes of the three mutant p62 proteins is significantly reduced.

Assessing the Biochemistry of Kindlin-3 In Human Platelets

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2011-2011
Author(s):  
Craig N. Streu ◽  
David Thomas Moore ◽  
Paul C. Billings ◽  
Patrik Nygren ◽  
Karen P. Fong ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Transmembrane Domain ◽  
Conflicts Of Interest ◽  
Short Form ◽  
Cytoplasmic Domain ◽  
Human Platelets ◽  
Resonance Spectroscopy ◽  
Ph Domain ◽  
Biochemical Basis ◽  
And Function

Abstract Abstract 2011 Although both talin and kindlin-3 binding to the β3 cytoplasmic domain are required for agonist-induced αIIbβ3 activation in platelets, the biochemical basis for this dual requirement is not clear. Recent NMR and hydrogen-deuterium exchange studies of disulfide-stabilized complexes containing the full cytoplasmic domains of αIIb and β3 dispersed in detergent micelles or lipid bilayers revealed that the β3 cytoplasmic domain consists of three helices: a stable proximal helix contiguous with the transmembrane domain and two distal dynamic amphiphilic helices whose fluctuations allow interaction of the helices with lipid bilayers or cytoplasmic proteins. These results suggest a cooperative model for talin and kindlin-3 binding to β3 with the talin and kindlin-3 binding sites kinetically- and thermodynamically-linked. Whether there is a preferred temporal sequence for kindlin-3 versus talin binding to β3 during physiological αIIbβ3 activation in platelets is not known, but the greater mobility of the kindlin-3 binding site suggests it might have a kinetic advantage over talin, assuming both are present in appropriate forms for binding. Much is known about the structure and function of talin, but substantially less is known about kindlin-3 in part because it has not been possible to express the complete molecule in bacterial expression systems. To address this issue, we have examined kindlin-3 expression and function in human platelets. Two kindlin-3 isoforms have been identified, a long form (Mr ∼ 76 kDa; accession: NM_178443) and a short form (Mr ∼75 kDa accession: NM_031471), that differ by the presence of 4 residues (RIPR; residues 360–363) in the PH domain of the long isoform. Using highly purified platelet and leukocyte RNA and RT PCR, we found that kindlin-3 expressed in platelets and leukocytes consist almost entirely of the 663 amino acid short isoform. Kindlin-3 present in platelet extracts is functionally active and spontaneously binds to the β3 cytoplasmic domain in pull down assays. Moreover, because kindlin-3 binding is abrogated by the β3 mutations S752P and T759A, but not T759F, the in vitro binding of kindlin-3 does not appear to be phosphorylation dependent. Further, surface plasmon resonance spectroscopy suggests that the PH domain of kindlin-3 partially drives membrane binding in the presence of phospholipids. Treatment of washed human platelets with the PAR1-activating peptide TRAP-6 (SFLLRNP) resulted in the rapid incorporation of kindlin-3 present in the platelet cytosol into the platelet cytoskeleton. We also found that kindlin-3 is present in dense fractions when platelets extracts were fractionated on sucrose gradients. Likewise, immunofluorescent images of platelets adherent to fibrinogen and platelet immuno-electron microscopy detected the presence of kindlin-3 in vesicular structures. Finally, using out-dated human platelets as starting material, we purified kindlin-3 to near homogeneity by the sequential use of ion-exchange and gel-filtration chromatography. These results provide a foundation for understanding the unique role that kindlin-3 plays in regulating the activity of platelet αIIbβ3. Disclosures: No relevant conflicts of interest to declare.

Subunit-dependent Inhibition of Human Neuronal Nicotinic Acetylcholine Receptors and Other Ligand-gated Ion Channels by Dissociative Anesthetics Ketamine and Dizocilpine

2000 ◽  
Vol 92 (4) ◽  
pp. 1144-1153 ◽  
Author(s):  
Tomohiro Yamakura ◽  
Laura E. Chavez-Noriega ◽  
R. Adron Harris
Keyword(s):  
Ion Channels ◽  
Nmda Receptors ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Dependent Manner ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Glycine Receptors ◽  
Nicotinic Acetylcholine ◽  
Dissociative Anesthetics ◽  
Gated Ion Channels

Unlabelled Background The neuronal mechanisms responsible for dissociative anesthesia remain controversial. N-methyl-D-aspartate (NMDA) receptors are inhibited by ketamine and related drugs at concentrations lower than those required for anesthetic effects. Thus, the authors studied whether ligand-gated ion channels other than NMDA receptors might display a sensitivity to ketamine and dizocilpine that is consistent with concentrations required for anesthesia. Methods Heteromeric human neuronal nicotinic acetylcholine receptors (hnAChR channels alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2 and alpha4beta4), 5-hydroxytryptamine3 (5-HT3), alpha1beta2gamma2S gamma-aminobutyric acid type A (GABAA) and alpha1 glycine receptors were expressed in Xenopus oocytes, and effects of ketamine and dizocilpine were studied using the two-electrode voltage-clamp technique. Results Both ketamine and dizocilpine inhibited hnAChRs in a noncompetitive and voltage-dependent manner. Receptors containing beta1 subunits were more sensitive to ketamine and dizocilpine than those containing beta2 subunits. The inhibitor concentration for half-maximal response (IC50) values for ketamine of hnAChRs composed of beta4 subunits were 9.5-29 microM, whereas those of beta2 subunits were 50-92 microM. Conversely, 5-HT3 receptors were inhibited only by concentrations of ketamine and dizocilpine higher than the anesthetic concentrations. This inhibition was mixed (competitive/noncompetitive). GABAA and glycine receptors were very resistant to dissociative anesthetics. Conclusions Human nAChRs are inhibited by ketamine and dizocilpine at concentrations possibly achieved in vivo during anesthesia in a subunit-dependent manner, with beta subunits being more critical than alpha subunits. Conversely, 5-HT3, GABAA, and glycine receptors were relatively insensitive to dissociative anesthetics.

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