Optically activated, customizable, excitable cells

Genetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca2+ and voltage sensors are available to study the signaling behavior of entire neural circuits. There is a pressing need for improved sensors, but improving them is challenging because testing them involves a low throughput, labor-intensive processes. Our goal was to create synthetic, excitable cells that can be activated with brief pulses of blue light and serve as a medium throughput platform for screening the next generation of sensors. In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP (Stierl M et al. 2011). In turn, the cAMP opens a cAMP-gated ion channel. This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we add the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. The result is a highly reproducible, medium-throughput, live cell system that can be used to screen voltage and Ca2+ sensors.

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Optically Activated, Customizable, Excitable Cells - A Kuhl Platform for Evolving Next Gen Biosensors

10.1101/2020.02.03.932426 ◽

2020 ◽

Author(s):

Merrilee Thomas ◽

Thom Hughes

Keyword(s):

Cyclic Nucleotide ◽

Connexin 43 ◽

Cell System ◽

Live Cell ◽

Hek293 Cells ◽

Modular System ◽

Intracellular Camp ◽

Excitable Cells ◽

Gated Channel ◽

Complex Signaling

AbstractGenetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca2+ and voltage sensors are available to study the signaling behavior of entire neural circuits. There is a pressing need for improved sensors to properly interrogate these systems. Improving them is challenging because testing them involves low throughput, labor-intensive processes. Our goal was to create a live cell system in HEK293 cells that use a simple, reproducible, optogenetic process for testing prototypes of genetically encoded biosensors.In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP [1]. In turn, the cAMP opens a cAMP gated ion channel (olfactory cyclic nucleotide-gated channel, CNG, or the hyperpolarization-activated cyclic nucleotide-gated channel, HCN2). This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we added the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. This is a modular system in which the types of channels, and their relative amounts, can be tuned to produce the cellular behavior that is crucial for screening biosensors. The result is a highly reproducible, high-throughput live cell system that can be used to screen voltage and Ca2+ sensors in multiple fluorescent wavelengths simultaneously.

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The inwardly rectifying potassium channel subunit Kir2.2v (KCNJN1) maps to 17p11.2→p11.1

Cytogenetic and Genome Research ◽

10.1159/000134688 ◽

1997 ◽

Vol 79 (1-2) ◽

pp. 85-87 ◽

Cited By ~ 5

Author(s):

N. Namba ◽

R. Mori ◽

H. Tanaka ◽

I. Kondo ◽

K. Narahara ◽

...

Keyword(s):

Potassium Channel ◽

Inwardly Rectifying Potassium Channel ◽

Inwardly Rectifying

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Modulation by histamine of an inwardly rectifying potassium channel in human endothelial cells.

The Journal of Physiology ◽

10.1113/jphysiol.1993.sp019951 ◽

1993 ◽

Vol 472 (1) ◽

pp. 359-371 ◽

Cited By ~ 29

Author(s):

B Nilius ◽

G Schwarz ◽

G Droogmans

Keyword(s):

Endothelial Cells ◽

Potassium Channel ◽

Human Endothelial Cells ◽

Inwardly Rectifying Potassium Channel ◽

Inwardly Rectifying

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The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00087.2011 ◽

2012 ◽

Vol 302 (3) ◽

pp. G397-G405 ◽

Cited By ~ 36

Author(s):

A. Chin ◽

B. Svejda ◽

B. I. Gustafsson ◽

A. B. Granlund ◽

A. K. Sandvik ◽

...

Keyword(s):

Mechanical Stimulation ◽

Tryptophan Hydroxylase ◽

Cell Function ◽

Cell System ◽

Receptor Expression ◽

Intracellular Camp ◽

Serotonin Secretion ◽

A2b Receptor ◽

Ec Cells ◽

Inflammatory Bowel

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98–99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10−6 M; IC50 = 3.7 × 10−8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT1 (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT1 transcription was regulated by PKA/MAPK and PI3K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.

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