Correct way to determine single-channel conductance from acetycholine receptors

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Exedore
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Correct way to determine single-channel conductance from acetycholine receptors

Hi, i'm looking for the best way to determine the single-channel conductance for a cys-loop receptor. I'm working on the AChR in outside-out and cell-attached configurations, but i'm trying to understand which is the most appropiate method to determine the single-channel conductance and how can i do it... by the noise-response method or single-channel amplitude ramp???
I hope my question be clear...
thanks in advance

Fraser Moss
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Common determinants of single channel conductance within the large cytoplasmic loop of 5-hydroxytryptamine type 3 and alpha4beta2 nicotinic acetylcholine receptors.
Hales TG, Dunlop JI, Deeb TZ, Carland JE, Kelley SP, Lambert JJ, Peters JA.
J Biol Chem. 2006 Mar 24;281(12):8062-71.
Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (gamma) below the resolution of single channel recording (966 +/- 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range gamma values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4'), 436 (0'), and 440 (4') residues by 5-HT3B (-4'Gln, 0'Asp, and 4'Ala) residues increases gamma to 36.5 +/- 1.0 pS. The 0' residue makes the most substantial contribution to gamma of the 5-HT3AR. Replacement of 0'Arg by aspartate, glutamate (alpha7 nAChR subunit MA 0'), or glutamine (beta2 subunit MA 0') increases gamma to the resolvable range (>6 pS). By contrast, replacement of 0'Arg by phenylalanine (alpha4 subunit MA 0') reduced gamma to 416 +/- 107 fS. In reciprocal experiments with alpha4beta2 nAChRs (gamma = 31.3 +/- 0.8 pS), replacement of MA 0' residues by arginine in alpha4beta2(Q443R) and alpha4(F588R)beta2 reduced gamma slightly. By contrast, the gamma of double mutant alpha4(F588R)beta2(Q443R) was halved. The MA -4' and 4' residues also influenced gamma of 5-HT3ARs. Replacement of nAChR alpha4 or beta2 MA 4' residues by arginine made current density negligible. By contrast, replacement of both -4' residues by arginine produced functional nAChRs with substantially reduced gamma (11.4 +/- 0.5 pS). Homology models of the 5-HT3A and alpha4beta2 nAChRs against Torpedo nAChR revealed MA -4', 0', and 4' residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.
PMID: 16407231
Molecular tuning of fast gating in pentameric ligand-gated ion channels.

Proc Natl Acad Sci U S A. 2005 December 13; 102(50): 18207–18212.
 

Grutter T, de Carvalho LP, Dufresne V, Taly A, Edelstein SJ, Changeux JP.
Neurotransmitters such as acetylcholine (ACh) and glycine mediate fast synaptic neurotransmission by activating pentameric ligand-gated ion channels (LGICs). These receptors are allosteric transmembrane proteins that rapidly convert chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with an extracellular agonist-binding domain (ECD), triggering a tertiary/quaternary conformational change in the protein that results in the fast opening of an ion pore domain (IPD). However, the molecular mechanism that determines the fast opening of LGICs remains elusive. Here, we show by combining whole-cell and single-channel recordings of recombinant chimeras between the ECD of alpha7 nicotinic receptor (nAChR) and the IPD of the glycine receptor (GlyR) that only two GlyR amino acid residues of loop 7 (Cys-loop) from the ECD and at most five alpha7 nAChR amino acid residues of the M2-M3 loop (2-3L) from the IPD control the fast activation rates of the alpha7/Gly chimera and WT GlyR. Mutual interactions of these residues at a critical pivot point between the agonist-binding site and the ion channel fine-tune the intrinsic opening and closing rates of the receptor through stabilization of the transition state of activation. These data provide a structural basis for the fast opening of pentameric LGICs.

 

Fraser Moss
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If you institution is

If you institution is subscribed this may help you too
http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-492-6_3
Single-Channel Recording
By:
David J.A. Wyllie

Joking90
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 Hi I'm a student at the

 Hi I'm a student at the University of Dundee and Ninewell's hospital. I'm studying the chimera alpha(7)-5-HT(3A) and how this relates subunit protein to function. As the alpha(7) holds the N-terminal I understand the effects of ACh, alpha-bungarotoxin and IC50 however I'm afraid I don't quite follow how single channel conductance (gamma) can be determined by the 5HT(3A) since this makes up the helixes of the TM1 through 4. I'm sure its simple but could someone explain why this happens? Thank you!