Novel structural determinants of single-channel conductance in nicotinic acetylcholine and 5-hydroxytryptamine type-3 receptors

2006 ◽  
Vol 34 (5) ◽  
pp. 882-886 ◽  
Author(s):  
J.A. Peters ◽  
J.E. Carland ◽  
M.A. Cooper ◽  
M.R. Livesey ◽  
T.Z. Deeb ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Single Channel ◽  
Ion Selectivity ◽  
Atomic Scale ◽  
Scale Model ◽  
Amino Acid Residues ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Structural Determinants ◽  
Type 3

Nicotinic ACh (acetylcholine) and 5-HT3 (5-hydroxytryptamine type-3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Numerous lines of evidence indicate that the channel lining domain of such receptors is formed by the α-helical M2 domain (second transmembrane domain) contributed by each of five subunits present within the receptor complex. Specific amino acid residues within the M2 domain have accordingly been demonstrated to influence both single-channel conductance (γ) and ion selectivity. However, it is now clear from work performed on the homomeric 5-HT3A receptor, heteromeric 5-HT3A/5-HT3B receptor and 5-HT3A/5-HT3B receptor subunit chimaeric constructs that an additional major determinant of γ resides within a cytoplasmic domain of the receptor termed the MA-stretch (membrane-associated stretch). The MA-stretch, within the M3–M4 loop, is not traditionally thought to be implicated in ion permeation and selection. Here, we describe how such observations extend to a representative neuronal nicotinic ACh receptor composed of α4 and β2 subunits and, by inference, probably other members of the Cys-loop family. In addition, we will attempt to interpret our results within the context of a recently developed atomic scale model of the nicotinic ACh receptor of Torpedo marmorata (marbled electric ray).

scholarly journals Rings of Charge within the Extracellular Vestibule Influence Ion Permeation of the 5-HT3A Receptor

2011 ◽  
Vol 286 (18) ◽  
pp. 16008-16017 ◽  
Author(s):  
Matthew R. Livesey ◽  
Michelle A. Cooper ◽  
Jeremy J. Lambert ◽  
John A. Peters
Keyword(s):  
Transmembrane Domain ◽  
Single Channel ◽  
Ion Selectivity ◽  
Amino Acid Residues ◽  
Charge Selectivity ◽  
Outward Currents ◽  
Flanking Sequences ◽  
Single Channel Currents ◽  
Key Residues ◽  
Channel Currents

The determinants of single channel conductance (γ) and ion selectivity within eukaryotic pentameric ligand-gated ion channels have traditionally been ascribed to amino acid residues within the second transmembrane domain and flanking sequences of their component subunits. However, recent evidence suggests that γ is additionally controlled by residues within the intracellular and extracellular domains. We examined the influence of two anionic residues (Asp113 and Asp127) within the extracellular vestibule of a high conductance human mutant 5-hydroxytryptamine type-3A (5-HT3A) receptor (5-HT3A(QDA)) upon γ, modulation of the latter by extracellular Ca2+, and the permeability of Ca2+ with respect to Cs+ (PCa/PCs). Mutations neutralizing (Asp → Asn), or reversing (Asp → Lys), charge at the 113 locus decreased inward γ by 46 and 58%, respectively, but outward currents were unaffected. The D127N mutation decreased inward γ by 82% and also suppressed outward currents, whereas the D127K mutation caused loss of observable single channel currents. The forgoing mutations, except for D127K, which could not be evaluated, ameliorated suppression of inwardly directed single channel currents by extracellular Ca2+. The PCa/PCs of 3.8 previously reported for the 5-HT3A(QDA) construct was reduced to 0.13 and 0.06 by the D127N and D127K mutations, respectively, with lesser, but clearly significant, effects caused by the D113N (1.04) and D113K (0.60) substitutions. Charge selectivity between monovalent cations and anions (PNa/PCl) was unaffected by any of the mutations examined. The data identify two key residues in the extracellular vestibule of the 5-HT3A receptor that markedly influence γ, PCa/PCs, and additionally the suppression of γ by Ca2+.

Interaction of Selective Amino Acid Residues of KCa Channels with Carbon Monoxide

2003 ◽  
Vol 228 (5) ◽  
pp. 474-480 ◽  
Author(s):  
Rui Wang ◽  
Lingyun Wu
Keyword(s):  
Smooth Muscle ◽  
Amino Acid ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Amino Acid Residues ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Channel Proteins

The activation of big-conductance KCa channels in vascular smooth muscle cells by carbon monoxide (CO) has been demonstrated previously. One specific target of CO on KCa channel proteins is the histidine residue. The roles of other amino acid residues on the functionality of KCa channels, as well as their reactions to CO, have been unclear. In the present study, the cell-free single channel recording technique was used to investigate the chemical modification of KCa channels by CO and other chemical agents. The modification of negatively charged carboxyl groups and the ε-amino group of lysine did not affect the open probability, but decreased single-channel conductance of KCa channels. When sulfhydryl groups of cysteine were modified with N-ethylmaleimide, the open probability of KCa channels was decreased, but single-channel conductance was not affected. None of the above chemical modifications affected the CO-induced increase in the open probability of KCa channels. However, N-ethylmaleimide treatment reduced the stimulatory effect of nitric oxide (NO) on KCa channels. Finally, pretreatment of smooth muscle cells with NO abolished the effects of subsequently applied CO on KCa channel proteins. Our study demonstrates that CO and NO acted on different amino acid residues of KCa channel proteins. The interaction of CO and NO determines the functional status of KCa channels in vascular smooth muscle cells

The 5-hydroxytryptamine type 3 (5-HT3) receptor reveals a novel determinant of single-channel conductance

2004 ◽  
Vol 32 (3) ◽  
pp. 547-552 ◽  
Author(s):  
J.A. Peters ◽  
S.P. Kelley ◽  
J.I. Dunlop ◽  
E.F. Kirkness ◽  
T.G. Hales ◽  
...  
Keyword(s):  
Ion Channels ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Single Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Nicotinic Acetylcholine ◽  
Ultrastructural Studies ◽  
Arginine Residues ◽  
Type 3

5-HT3 (5-hydroxytryptamine type 3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Two 5-HT3 receptor subunits, 5-HT3A and 5-HT3B, have been characterized in detail, although additional putative 5-HT3 subunit genes (HTR3C, HTR3D and HTR3E) have recently been reported. 5-HT3 receptors function as homopentameric assemblies of the 5-HT3 subunit, or heteropentamers of 5-HT3A and 5-HT3B subunits of unknown stoichiometry. The single-channel conductances of human recombinant homomeric and heteromeric 5-HT3 receptors are markedly different, being <1 and approx. 16 pS respectively. Paradoxically, from the results of studies performed on the closely related nicotinic acetylcholine receptor, the channel-lining M2 domain of the 5-HT3A subunit is predicted to enhance cation conduction, whereas that of the 5-HT3B subunit would not. The present study describes a novel determinant of single-channel conductance, outwith the M2 domain, which accounts for this anomaly. Utilizing a panel of chimaeric 5-HT3A and 5-HT3B subunits, a profound determinant of single-channel conductance was traced to a putative amphipathic helix (the ‘HA stretch’) within the large cytoplasmic loop of the receptor. Replacement of three arginine residues (R432, R436 and R440) unique to the HA stretch of the 5-HT3A subunit with the aligned residues (Q395, D399 and A403) of the 5-HT3B subunit increased the single-channel conductance 28-fold. Significantly, from ultrastructural studies of the Torpedo nicotinic acetylcholine receptor, the key residues may be components of narrow openings within the inner vestibule of the channel, located in the cytoplasm, which contribute to the permeation pathway. Our findings indicate an important and hitherto unappreciated function for the HA stretch in the Cys-loop family of transmitter-gated ion channels.

scholarly journals Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2Nicotinic Acetylcholine Receptors

2006 ◽  
Vol 281 (12) ◽  
pp. 8062-8071 ◽  
Author(s):  
Tim G. Hales ◽  
James I. Dunlop ◽  
Tarek Z. Deeb ◽  
Jane E. Carland ◽  
Stephen P. Kelley ◽  
...  
Keyword(s):  
Acetylcholine Receptors ◽  
Single Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Cytoplasmic Loop ◽  
Type 3
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