TY - JOUR
T1 - Nicotinic receptor fourth transmembrane domain
T2 - Hydrogen bonding by conserved threonine contributes to channel gating kinetics
AU - Bouzat, Cecilia
AU - Barrantes, Francisco
AU - Sine, Steven
PY - 2000/5
Y1 - 2000/5
N2 - The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association width the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated αT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of αT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of αT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.
AB - The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association width the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated αT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of αT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of αT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.
KW - Fourth transmembrane domain
KW - Hydrogen bond
KW - Kinetic analysis
KW - Nicotinic acetylcholine receptor channel gating
KW - Patch clamp
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U2 - 10.1085/jgp.115.5.663
DO - 10.1085/jgp.115.5.663
M3 - Article
C2 - 10779322
AN - SCOPUS:0034059664
SN - 0022-1295
VL - 115
SP - 663
EP - 671
JO - Journal of General Physiology
JF - Journal of General Physiology
IS - 5
ER -