@article {67848, title = {Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.}, journal = {eLife}, volume = {11}, year = {2022}, month = {2022 Jan 04}, abstract = {

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ({\textquoteright}iDrugSnFRs{\textquoteright}) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by \> 30 fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

}, issn = {2050-084X}, doi = {10.7554/eLife.74648}, author = {Nichols, Aaron L and Blumenfeld, Zack and Fan, ChengCheng and Luebbert, Laura and Blom, Annet Em and Cohen, Bruce N and Marvin, Jonathan S and Borden, Philip M and Kim, Charlene H and Muthusamy, Anand K and Shivange, Amol V and Knox, Hailey J and Campello, Hugo Rego and Wang, Jonathan H and Dougherty, Dennis A and Looger, Loren L and Gallagher, Timothy and Rees, Douglas C and Lester, Henry A} } @article {65604, title = {Nanoscopic visualization of restricted nonvolume cholinergic and monoaminergic transmission with genetically encoded sensors.}, journal = {Nano Letters}, volume = {20}, year = {2020}, month = {2020 Jun}, pages = {4073-83}, abstract = {

How neuromodulatory transmitters diffuse into the extracellular space remains an unsolved fundamental biological question, despite wide acceptance of the volume transmission model. Here, we report development of a method combining genetically encoded fluorescent sensors with high-resolution imaging and analysis algorithms which permits the first direct visualization of neuromodulatory transmitter diffusion at various neuronal and non-neuronal cells. Our analysis reveals that acetylcholine and monoamines diffuse at individual release sites with a spread length constant of \~{}0.75 μm. These transmitters employ varied numbers of release sites, and when spatially close-packed release sites coactivate they can spillover into larger subcellular areas. Our data indicate spatially restricted (i.e., nonvolume) neuromodulatory transmission to be a prominent intercellular communication mode, reshaping current thinking of control and precision of neuromodulation crucial for understanding behaviors and diseases.

}, issn = {1530-6992}, doi = {10.1021/acs.nanolett.9b04877}, author = {Zhu, Paula K and Zheng, W Sharon and Zhang, Peng and Jing, Miao and Borden, Philip M and Ali, Farhan and Guo, Kaiming and Feng, Jiesi and Marvin, Jonathan S and Wang, Yali and Wan, Jinxia and Gan, Li and Kwan, Alex C and Lin, Li and Looger, Loren L and Li, Yulong and Zhang, Yajun} } @article {65242, title = {Biosensors show the pharmacokinetics of S-Ketamine in the endoplasmic reticulum.}, journal = {Frontiers in Cellular Neuroscience}, volume = {13}, year = {2019}, month = {2019 Nov 12}, pages = {499}, abstract = {

The target for the "rapid" (\<24 h) antidepressant effects of S-ketamine is unknown, vitiating programs to rationally develop more effective rapid antidepressants. To describe a drug{\textquoteright}s target, one must first understand the compartments entered by the drug, at all levels-the organ, the cell, and the organelle. We have, therefore, developed molecular tools to measure the subcellular, organellar pharmacokinetics of S-ketamine. The tools are genetically encoded intensity-based S-ketamine-sensing fluorescent reporters, iSKetSnFR1 and iSKetSnFR2. In solution, these biosensors respond to S-ketamine with a sensitivity, S-slope = delta(F/F)/(delta[S-ketamine]) of 0.23 and 1.9/μM, respectively. The iSKetSnFR2 construct allows measurements at \<0.3 μM S-ketamine. The iSKetSnFR1 and iSKetSnFR2 biosensors display \>100-fold selectivity over other ligands tested, including R-ketamine. We targeted each of the sensors to either the plasma membrane (PM) or the endoplasmic reticulum (ER). Measurements on these biosensors expressed in Neuro2a cells and in human dopaminergic neurons differentiated from induced pluripotent stem cells (iPSCs) show that S-ketamine enters the ER within a few seconds after appearing in the external solution near the PM, then leaves as rapidly after S-ketamine is removed from the extracellular solution. In cells, S-slopes for the ER and PM-targeted sensors differ by \<2-fold, indicating that the ER [S-ketamine] is less than 2-fold different from the extracellular [S-ketamine]. Organelles represent potential compartments for the engagement of S-ketamine with its antidepressant target, and potential S-ketamine targets include organellar ion channels, receptors, and transporters.

}, issn = {1662-5102}, doi = {10.3389/fncel.2019.00499}, author = {Bera, Kallol and Kamajaya, Aron and Shivange, Amol V and Muthusamy, Anand K and Nichols, Aaron L and Borden, Philip M and Grant, Stephen and Jeon, Janice and Lin, Elaine and Bishara, Ishak and Chin, Theodore M and Cohen, Bruce N and Kim, Charlene H and Unger, Elizabeth K and Tian, Lin and Marvin, Jonathan S and Looger, Loren L and Lester, Henry A} } @article {64546, title = {Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors.}, journal = {The Journal of General Physiology}, year = {2019}, month = {2019 Feb 04}, abstract = {

Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an "inside-out" pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] \<1 {\textmu}M, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell-derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as \~{}10 nM. Such concentrations would persist during the 12 h of a typical smoker{\textquoteright}s day, continually activating the inside-out pathway by \>75\%. Reducing nicotine intake by 10-fold decreases activation to \~{}20\%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

}, issn = {1540-7748}, doi = {10.1085/jgp.201812201}, author = {Shivange, Amol V and Borden, Philip M and Muthusamy, Anand K and Nichols, Aaron L and Bera, Kallol and Bao, Huan and Bishara, Ishak and Jeon, Janice and Mulcahy, Matthew J and Cohen, Bruce and O{\textquoteright}Riordan, Saidhbhe L and Kim, Charlene and Dougherty, Dennis A and Chapman, Edwin R and Marvin, Jonathan and Looger, Loren and Lester, Henry A} } @article {64783, title = {Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors.}, journal = {The Journal of General Physiology}, volume = {151}, year = {2019}, month = {2019 Feb 04}, pages = {738-57}, abstract = {

Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an "inside-out" pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] \<1 {\textmu}M, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell-derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as \~{}10 nM. Such concentrations would persist during the 12 h of a typical smoker{\textquoteright}s day, continually activating the inside-out pathway by \>75\%. Reducing nicotine intake by 10-fold decreases activation to \~{}20\%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

}, issn = {1540-7748}, doi = {10.1085/jgp.201812201}, author = {Shivange, Amol V and Borden, Philip M and Muthusamy, Anand K and Nichols, Aaron L and Bera, Kallol and Bao, Huan and Bishara, Ishak and Jeon, Janice and Mulcahy, Matthew J and Cohen, Bruce and O{\textquoteright}Riordan, Saidhbhe L and Kim, Charlene and Dougherty, Dennis A and Chapman, Edwin R and Marvin, Jonathan and Looger, Loren and Lester, Henry A} } @article {64796, title = {A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP.}, journal = {Nature Communications}, volume = {10}, year = {2019}, month = {2019 Feb 12}, pages = {711}, abstract = {

Adenosine 5{\textquoteright} triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of FF-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR responds to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-08441-5}, author = {Lobas, Mark A and Tao, Rongkun and Nagai, Jun and Kronschl{\"a}ger, Mira T and Borden, Philip M and Marvin, Jonathan S and Looger, Loren L and Khakh, Baljit S} }