Highly Parallelized, Multicolor Optogenetic Recordings of Cellular Activity for Therapeutic Discovery Applications in Ion Channels and Disease-Associated Excitable Cells.

Optogenetic assays provide a flexible, scalable, and information rich approach to probe compound effects for ion channel drug targets in both heterologous expression systems and associated disease relevant cell types. Despite the potential utility and growing adoption of optogenetics, there remains a critical need for compatible platform technologies with the speed, sensitivity, and throughput to enable their application to broader drug screening applications. To address this challenge, we developed the SwarmTM, a custom designed optical instrument for highly parallelized, multicolor measurements in excitable cells, simultaneously recording changes in voltage and calcium activities at high temporal resolution under optical stimulation. The compact design featuring high power LEDs, large numerical aperture optics, and fast photodiode detection enables all-optical individual well readout of 24-wells simultaneously from multi-well plates while maintaining sufficient temporal resolution to probe millisecond response dynamics. The Swarm delivers variable intensity blue-light optogenetic stimulation to enable membrane depolarization and red or lime-light excitation to enable fluorescence detection of the resulting changes in membrane potential or calcium levels, respectively. The Swarm can screen ~10,000 wells/day in 384-well format, probing complex pharmacological interactions via a wide array of stimulation protocols. To evaluate the Swarm screening system, we optimized a series of heterologous optogenetic spiking HEK293 cell assays for several voltage-gated sodium channel subtypes including Nav1.2, Nav1.5, and Nav1.7. The Swarm was able to record pseudo-action potentials stably across all 24 objectives and provided pharmacological characterization of diverse sodium channel blockers. We performed a Nav1.7 screen of 200,000 small molecules in a 384-well plate format with all 560 plates reaching a Z' > 0.5. As a demonstration of the versatility of the Swarm, we also developed an assay measuring cardiac action potential and calcium waveform properties simultaneously under paced conditions using human induced pluripotent stem (iPS) cell-derived cardiomyocytes as an additional counter screen for cardiac toxicity. In summary, the Swarm is a novel high-throughput all-optical system capable of collecting information-dense data from optogenetic assays in both heterologous and iPS cell-derived models, which can be leveraged to drive diverse therapeutic discovery programs for nervous system disorders and other disease areas involving excitable cells.

Wild edible plants collected by Hani from terraced rice paddy agroecosystem in Honghe Prefecture, Yunnan, China.

BACKGROUND: The Hani people in the Honghe Prefecture of Southeastern Yunnan, China, have practiced terraced rice paddy farming for more than 1300 years. These rice fields, combined with the surrounding forests and water systems, form a special agroecosystem that has attracted both tourists and scientists. For centuries, the local people have traditionally collected wild edible plants (WEP) from the agroecosystem, but this unique traditional practice in this area has never been reported. METHODS: Ethnobotanical fieldwork was conducted in four counties (Yuanyang, Honghe, Jinping, and Lüchun) between 2014 and 2019. Local self-identified Hani people (186) were interviewed, and information concerning local WEP species was obtained, documented, and analyzed. Plant samples and voucher specimens were collected for taxonomic identification. RESULTS: A total of 224 WEP species, belonging to 90 families and 170 genera, were recorded as used by the Hani people in Honghe. The most common WEP parts used include fruits, stems, and leaves, and the most common preparation methods include eating as a potherb (wild vegetable) and eating fresh. Some WEPs, like Phyllanthus emblica and Dioscorea subcalva, have unique preparation methods. The use-value (UV) and frequency of utilization index (FUI) of WEP species were analyzed. The 20 WEP species with the highest UV were noted as particularly important to the Hani people's daily life in Honghe. CONCLUSION: A large majority of these WEP species possess tremendous economic potential for future development. However, the diversity of WEP species, the associated traditional knowledge, and the broader agroecosystem are facing challenges such as biodiversity loss and pollution from chemical pesticides and fertilizers. This study may help local people to recognize the value of local WEP species and associated traditional knowledge, as well as provide ethnobotanical information for the future development of this tourism region.

Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels.

Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1-KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1-KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)4(KCNE1)n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or IKs, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether IKs is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments IKs current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)4(KCNE1)n channels are present as components of IKs and are pharmacologically distinct from KCNE-saturated KCNQ1-KCNE1 channels.