M4531

The drug solution was carboxygenated, and bath was applied through perfusion for at least 15 min before the recordings. We used 100 μM meclofenamic acid (MFA; M4531, Sigma-Aldrich) to block gap junctional coupling. In additional experiments (Supplementary Figures), the following drug concentrations were used (in μM): 50 6,7-dinitroquinoxaline-2,3-dione (DNQX, 0189, TOCRIS, Bristol, United Kingdom) and 50 DL-2-amino-5-phosphonopentanoic acid sodium salt (DL-AP5, 3693, TOCRIS) to block ionotrophic glutamate receptors and 50 1,2,5,6-tetrahydropyridin-4-yl methylphosphinic acid (TPMPA, 1040, TOCRIS) and 25 6-Imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide (SR95531, 1262, TOCRIS) to block GABA receptors.

Where specified, any bleached visual pigment was regenerated with the artificial chromophore analog 9-cis-Retinal. Stock solutions of 9-cis-Retinal (R5754; Sigma–Aldrich) in ethanol (100 mM) were prepared in darkness and stored at −80°C. On the day of the experiment, an aliquot was thawed and diluted to a final concentration of 100 µM in Ames' medium integrated with 1% wt/vol fatty acid-free bovine serum albumin (A8806; Sigma–Aldrich), an effective solubilizing and protective agent (Li et al., 1999 (link)). This solution was delivered to the preparation directly in the recording chamber, without modifying flow rate or temperature, for 20–25 min followed by washout. A pharmacological blockade of gap junctions was attempted with meclofenamic acid (MFA; M4531, Sigma–Aldrich) and 2-aminoethyl diphenylborinate (2-APB; D9754, Sigma–Aldrich). The I_h current was blocked with 4-Ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD7288; cat. no. 1000, Tocris, United Kingdom).

To block the C×43 function, GFBLs were seeded on 6-well plates (42,000 cells/cm²) in their normal growth medium. At day 2 when cultures became confluent, culture medium was replaced with serum-free medium. At day 3, cells were treated with 150 μM of Gap27 (SRPTEKTIFII; Biomatik, Cambridge, ON, Canada) [3 (link),48 (link),49 (link)], equal molar amount of Gap26 (VCYDKSFPISHVR) [3 (link),48 (link)–50 (link)], or corresponding scrambled Gap27 (TFEPDRISITK) [33 (link)] or Gap26 (YSIVCKPHVFDRS) [50 (link)] control peptides, respectively, for up to 24 h before total RNA isolation or collection of cell lysates/conditioned medium for Western blotting. In a set of experiments, cells were treated with increasing concentrations (25, 50, 75, 100 μM) of meclofenamic acid (MFA; M4531, Sigma-Aldrich, St. Louis, MO, USA), a pharmacological connexin inhibitor [51 (link)], or corresponding amount of MFA diluent (dH₂O) for 24 h before RNA isolation.