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Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor

Philipson, Kenneth D. and Gallivan, Justin P. and Brandt, Gabriel S. and Dougherty, Dennis A. and Lester, Henry A. (2001) Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor. American Journal of Physiology. Cell Physiology, 281 (1). C195-C206. ISSN 0363-6143. doi:10.1152/ajpcell.2001.281.1.c195.

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The nonsense codon suppression technique was used to incorporate o-nitrobenzyl cysteine or o-nitrobenzyl tyrosine (caged Cys or Tyr) into the 9′ position of the M2 transmembrane segment of the γ-subunit of the muscle nicotinic ACh receptor expressed in Xenopusoocytes. The caged amino acids replaced an endogenous Leu residue that has been implicated in channel gating. ACh-induced current increased substantially after ultraviolet (UV) irradiation to remove the caging group. This represents the first successful incorporation of caged Cys into a protein in vivo and the first incorporation of caged amino acids within a transmembrane segment of a membrane protein. The bulky nitrobenzyl group does not prevent the synthesis, assembly, or trafficking of the ACh receptor. When side chains were decaged using 1-ms UV light flashes, the channels with caged Cys or caged Tyr responded with strikingly different kinetics. The increase in current upon photolysis of caged Cys was too rapid for resolution by the voltage-clamp circuit [time constant (τ) <10 ms], whereas the increase in current upon photolysis of caged Tyr was dominated by a phase with τ ∼500 ms. Apparently, the presence of a bulkyo-nitrobenzyl Tyr residue distorts the receptor into an abnormal conformation. Upon release of the caging group, the receptor relaxes, with τ ∼500 ms, into a conformation that allows the channel to open. Tyr at the 9′ position of the γ-subunit greatly increases the ability of ACh to block the channel by binding within the channel pore. This is manifested in two ways. 1) A “rebound,” or increase in current, occurs upon removal of ACh from the bathing medium; and 2) at ACh concentrations >400 μM, inward currents are decreased through the mutated channel. The ability to incorporate caged amino acids into proteins should have widespread utility.

Item Type:Article
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Dougherty, Dennis A.0000-0003-1464-2461
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2001 the American Physiological Society. Received 26 October 2000; Accepted 28 February 2001; Published online 1 July 2001; Published in print 1 July 2001. We thank Dr. B. Khakh for helpful discussions. This research was supported by grants from the National Institutes of Health (NS-11756, NS-34407, and HL-49101). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Subject Keywords:nicotinic acetylcholine receptor; ion channel; site-directed mutagenesis; nonsense suppression; o-nitrobenzyl tyrosine; o-nitrobenzyl cysteine
Issue or Number:1
Record Number:CaltechAUTHORS:20200506-103644435
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Official Citation:Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor Kenneth D. Philipson, Justin P. Gallivan, Gabriel S. Brandt, Dennis A. Dougherty, and Henry A. Lester American Journal of Physiology-Cell Physiology 2001 281:1, C195-C206; doi: 10.1152/ajpcell.2001.281.1.c195
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:103029
Deposited By: Tony Diaz
Deposited On:06 May 2020 17:43
Last Modified:16 Nov 2021 18:17

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