Using antibodies prepared against a unique region (exon 22-24) of rat K ϩ -Cl Ϫ cotransporter-2 (KCC2), we confirmed that the ϳ140-kDa KCC2 protein is exclusively expressed in rat brain, but in chicken, we observed strong reactivity not only with the ϳ140-kDa KCC2 protein in brain but also a slightly larger ϳ145-kDa protein in heart. In silico analysis showed that while exon 22 of KCC2 is unique to this isoform in therian mammals, it is retained in KCC2's closest paralog, KCC4, of lower vertebrates, including chicken. To eliminate potential crossreactivity with chicken KCC4, the antibodies were preadsorbed with blocking peptides prepared over the only two regions showing significant sequence identity to chicken KCC4. This completely eliminated antibody recognition of exogenously expressed chicken KCC4 but not of the ϳ145-kDa protein in chicken heart, indicating that chicken heart expresses KCC2. Real-time PCR confirmed robust KCC2 transcript expression in both chicken brain and heart. Chicken heart expressed predominantly the longer KCC2a splice variant consistent with the larger ϳ145-kDa protein in chicken heart. Immunofluorescence microscopy revealed prominent plasma membrane KCC2 labeling in chicken ventricular cardiomyocytes. We hypothesize that KCC2 is an important Cl Ϫ extrusion pathway in avian cardiomyocytes that counters channel-mediated Cl Ϫ loading during high heart rates with ␤-adrenergic stimulation. While KCC2 is absent from mammalian cardiomyocytes, understanding the role that the other KCC isoforms play in Cl Ϫ homeostasis of these cells represents a nascent area of research. chloride; homeostasis; KCC2; KCC4; avian; Cl Ϫ channels THE K ϩ -Cl

Fluorometric imaging plate reader membrane potential dye (FMP-Red-Dye) is a proprietary tool for basic discovery and high-throughput drug screening for G-protein-coupled receptors and ion channels. We optimized and validated this potentiometric probe to assay functional modulators of heterologous expressed GABA receptor (GABAR) isoforms (synaptic 132, extrasynaptic 43, and 3 homopentomers). High-resolution mass spectrometry identified FMP-Red-Dye as 5,5'-(1-propen-1-yl-3-ylidene)bis[1,3-dimethyl-2-thio-barbituric acid]. GABAR-expressing cells equilibrated with FMP-Red-Dye exhibited depolarized equilibrium membrane potentials compared with GABAR-null cells. The channel blockers picrotoxin, fipronil, and tetramethylenedisulfotetramine, and the competitive antagonist bicuculline reduced fluorescence near the levels in GABAR-null cells indicating that FMR-Red-Dye, a barbiturate derivative, activates GABAR-mediated outward Cl current in the absence of GABA. GABA caused concentration-dependent increases in fluorescence with rank order of potencies among GABAR isoforms consistent with results from voltage-clamp experiments (EC values for 43, 132, and homopentamers were 6 ± 1, 40 ± 11, and>18 mM, respectively), whereas GABAR-null cells were unresponsive. Neuroactive steroids (NAS) increased fluorescence of GABAR expressing cells in the absence of GABA and demonstrated positive allosteric modulation in the presence of GABA, whereas benzodiazepines only exhibited positive allosteric modulator (PAM) activity. Of 20 NAS tested, allopregnanolone, (3,5,20E)-3-hydroxy-13,24-cyclo-18-norcholan-20-ene-21-carbonitrile, eltanolone, 5-pregnan-3,21-diol-20-one, and ganaxolone showed the highest potency. The FMP-Red-Dye-based assay described here provides a sensitive and quantitative method of assessing the activity of GABAR agonists, antagonists, and PAMs on diverse GABAR isoforms. The assay has a wide range of applications, including screening for antiseizure agents and identifying channel blockers of interest to insecticide discovery or biosecurity.

Tetramethylenedisulfotetramine (tetramine; TETS), a potent convulsant, triggers abnormal electrical spike activity and synchronous Ca2+ oscillation (SCO) patterns in cultured neuronal networks by blocking GABAA receptors. Murine hippocampal neuronal/glia cocultures develop extensive dendritic connectivity between glutamatergic and GABAergic inputs and display two distinct SCO patterns when imaged with the Ca2+ indicator Fluo-4: Low Amplitude SCO Events (LASE) and High Amplitude SCO Events (HASE) that are dependent on TTX-sensitive network electrical spike activity (ESA). Acute TETS (3.0 µM) increased overall network SCO amplitude and decreased SCO frequency by stabilizing HASE and suppressing LASE while increasing ESA. In multielectrode arrays TETS also increased burst frequency and synchronicity. In the presence of TETS (3.0 µM), the clinically used anticonvulsive perampanel (0.1-3.0 µM), a noncompetitive AMPAR antagonist, suppressed all SCO activity, whereas the GABAA receptor potentiator midazolam (MDZ, 1.0-30 µM), the current standard of care, reciprocally suppressed HASE and stabilized LASE. The neuroactive steroid (NAS) allopregnanolone (ALLO; 0.1-3.0 µM) normalized TETS-triggered patterns by selectively suppressing HASE and increasing LASE, a pharmacological pattern distinct from its epimeric form eltanolone, ganaxolone, alphaxolone and XJ-42, which significantly potentiated TETS-triggered HASE in a biphasic manner. Cortisol failed to mitigate TETS-triggered patterns and at > 1 µM augmented them. Combinations of ALLO and MDZ were significantly more effective at normalizing TETS-triggered SCO patterns, ESA patterns, and more potently enhanced GABA-activated Cl- current, than either drug alone.

Bromopyrroles (BrPyr) are synthesized naturally by marine sponge symbionts and produced anthropogenically as byproducts of wastewater treatment. BrPyr interact with ryanodine receptors (RYRs) and sarco/endoplasmic reticulum (SR/ER) Ca2+-ATPase (SERCA). Influences of BrPyr on the neuronal network activity remain uncharted. BrPyr analogues with differing spectra of RYR/SERCA activities were tested using RYR-null or RYR1-expressing HEK293 and murine cortical neuronal/glial cocultures (NGCs) loaded with Fluo-4 to elucidate their mechanisms altering Ca2+ dynamics. The NGC electrical spike activity (ESA) was measured from NGCs plated on multielectrode arrays. Nanomolar tetrabromopyrrole (TBP, 1) potentiated caffeine-triggered Ca2+ release independent of extracellular [Ca2+] in RYR1-HEK293, whereas higher concentrations produce slow and sustained rise in cytoplasmic [Ca2+] independent of RYR1 expression. TBP, 2,3,5-tribromopyrrole (2), pyrrole (3), 2,3,4-tribromopyrrole (4), and ethyl 4-bromopyrrole-2-carboxylate (5) added acutely to NGC showed differential potency; rank order TBP (IC50 ≈ 220 nM) > 2 ≫ 5, whereas 3 and 4 were inactive at 10 μM. TBP >2 μM elicited sustained elevation of cytoplasmic [Ca2+] and loss of neuronal viability. TBP did not alter network ESA. BrPyr from marine and anthropogenic sources are ecological signaling molecules and emerging anthropogenic pollutants of concern to environmental and human health that potently alter ER Ca2+ dynamics and warrant further investigation in vivo.