Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors

Pantoja, Rigo; Rodriguez, Erik A.; Dibas, Mohammed; Dougherty, Dennis A.; Lester, Henry A.

doi:10.1016/j.bpj.2008.09.034

Cited by 65 publications

(52 citation statements)

References 65 publications

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“…It is also possible that SePhaChARNS could be manipulated by drugs that modify protein chaperones. Fluorescence-based analyses of nAChR assembly, trafficking, and stoichiometry are under way in the Caltech lab (73,(127)(128)(129). In principle, such analyses may be suitable for drug discovery relevant to the SePhaChARNS process.…”

Section: Conclusion: Implications For Drug Discoverymentioning

confidence: 99%

Nicotine is a Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry. Implications for Drug Discovery

Lester

Xia

Srinivasan

et al. 2009

AAPS J

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142

167

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Abstract. The acronym SePhaChARNS, for "selective pharmacological chaperoning of acetylcholine receptor number and stoichiometry," is introduced. We hypothesize that SePhaChARNS underlies classical observations that chronic exposure to nicotine causes "upregulation" of nicotinic receptors (nAChRs). If the hypothesis is proven, (1) SePhaChARNS is the molecular mechanism of the first step in neuroadaptation to chronic nicotine; and (2) nicotine addiction is partially a disease of excessive chaperoning. The chaperone is a pharmacological one, nicotine; and the chaperoned molecules are α4β2* nAChRs. SePhaChARNS may also underlie two inadvertent therapeutic effects of tobacco use: (1) the inverse correlation between tobacco use and Parkinson's disease; and (2) the suppression of seizures by nicotine in autosomal dominant nocturnal frontal lobe epilepsy. SePhaChARNS arises from the thermodynamics of pharmacological chaperoning: ligand binding, especially at subunit interfaces, stabilizes AChRs during assembly and maturation, and this stabilization is most pronounced for the highest-affinity subunit compositions, stoichiometries, and functional states of receptors. Several chemical and pharmacokinetic characteristics render exogenous nicotine a more potent pharmacological chaperone than endogenous acetylcholine. SePhaChARNS is modified by desensitized states of nAChRs, by acid trapping of nicotine in organelles, and by other aspects of proteostasis. SePhaChARNS is selective at the cellular, and possibly subcellular, levels because of variations in the detailed nAChR subunit composition, as well as in expression of auxiliary proteins such as lynx. One important implication of the SePhaChARNS hypothesis is that therapeutically relevant nicotinic receptor drugs could be discovered by studying events in intracellular compartments rather than exclusively at the surface membrane.

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Section: Conclusion: Implications For Drug Discoverymentioning

confidence: 99%

Nicotine is a Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry. Implications for Drug Discovery

Lester

Xia

Srinivasan

et al. 2009

AAPS J

Self Cite

142

167

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“…Previously, unnatural amino acids with various properties have been incorporated into ion channels by injecting chemically aminoacylated tRNAs into Xenopus oocytes (25-27) including a fUAA (28). Here, we used a fUAA to successfully carry out VCF experiments.…”

mentioning

confidence: 99%

Dynamics of internal pore opening in K _V channels probed by a fluorescent unnatural amino acid

Kalstrup

Blunck

2013

Proc. Natl. Acad. Sci. U.S.A.

102

126

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Atomic-scale models on the gating mechanism of voltage-gated potassium channels (Kv) are based on linear interpolations between static structures of their initial and final state derived from crystallography and molecular dynamics simulations, and, thus, lack dynamic structural information. The lack of information on dynamics and intermediate states makes it difficult to associate the structural with the dynamic functional data obtained with electrophysiology. Although voltage-clamp fluorometry fills this gap, it is limited to sites extracellularly accessible, when the key region for gating is located at the cytosolic side of the channels. Here, we solved this problem by performing voltage-clamp fluorometry with a fluorescent unnatural amino acid. By using an orthogonal tRNAsynthetase pair, the fluorescent unnatural amino acid was incorporated in the Shaker voltage-gated potassium channel at key regions that were previously inaccessible. Thus, we defined which parts act independently and which parts act cooperatively and found pore opening to occur in two sequential transitions.Anap | two-color VCF V oltage-gated potassium channels (K V ) are essential for generating action potentials in the central nervous system and, when defective, are linked to severe familial diseases including cardiac arrhythmias and epilepsy. The voltage-sensing domains (VSD) of K V channels (transmembrane helices S1-S4; Fig. 1A) undergo a major conformational change upon membrane depolarization driven by the positive charges in the S4, which finally leads to opening of the pore domain (transmembrane helices S5-S6). Based on the consensus on the closed (initial) and open (final) state structures (1-6), the gating movement has been predicted; the S4 helix is projected to slide upward and tilt with respect to the membrane normal, and this movement pushes the S4-S5 linker and the S6 helix inward and closes the ion-conducting pore. However, this projection relies on linear interpolations between the closed and open state and lacks any information on dynamics or intermediate states.As a result, the projected movement does not suffice to explain fundamental characteristics of voltage sensor and pore domain kinetics, detected as "gating" and "ionic" currents, respectively. Such functional electrophysiology measurements revealed that, first, at least one intermediate state has to exist during voltage sensor movement (7) and that, second, voltage sensor movement and pore opening do not occur simultaneously. Each channel consists of four voltage sensors controlling a single central pore. It is thought that the four voltage sensors activate independently, and only after all four have activated, the central pore opens cooperatively (8,9). This mechanism implies that the energy generated by the movement of the first three voltage sensors has to be "conserved" in the system and has to be released to the pore during opening (10). The linear interpolations between closed and open structures leave the basis of both cooperativity and energy conservation un...

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“…The Optosplit Emission GFP/RFP filter set (GFP: 525/30, DM 555, RFP: 598/40) and the microscope filter cube with dichroic (FF505/606-Di01-25×36) were from Semrock (Semrock, Rochester, NY). A Photometrics Cascade 650 charge-coupled device camera with 16-bit resolution (Princeton Instruments, Trenton, NJ) and an Andor IQ image capture and analysis software program (Andor Technology PLC, Belfast, Ireland) were used to capture images using a 300 ms integration time [10,15] . Colocalization of pmCherry and YFP images was visualized using Image J software with the colocalization finder plug-in [52] .…”

Section: Methodsmentioning

confidence: 99%

“…Alternatively, placing the FP in an intracellular domain might obviate these concerns; however, inserting it in this location presents other challenges including interruption of trafficking domains and phosphorylation sites, which could disrupt function [7] . Fortunately, several fluorescently tagged Cys-loop receptor subunits have been made with the FP inserted into the second, large intracellular loop (C2) between the third (M3) and fourth (M4) transmembrane domains; and these assembled receptors appear fully functional by several criteria [9,10,[13][14][15] . However this suite does not yet include a nAChR α7 subunit with a C2 FP fusion.…”

Section: Introductionmentioning

confidence: 99%

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

et al. 2009

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Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors. Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125 I-labeled α-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Results: Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC 50 values for the α7 nAChR-selective agonist, choline, and IC 50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion: nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

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Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors

Cited by 65 publications

References 65 publications

Nicotine is a Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry. Implications for Drug Discovery

Nicotine is a Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry. Implications for Drug Discovery

Dynamics of internal pore opening in K _V channels probed by a fluorescent unnatural amino acid

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

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Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors

Cited by 65 publications

References 65 publications

Nicotine is a Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry. Implications for Drug Discovery

Nicotine is a Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry. Implications for Drug Discovery

Dynamics of internal pore opening in K V channels probed by a fluorescent unnatural amino acid

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

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Dynamics of internal pore opening in K _V channels probed by a fluorescent unnatural amino acid