
Background:
Non-excitable cells show Vm fluctuations, but sub-mV imaging was limited; connexin/oncochannel roles were unclear.

Methods: rEstus2s was engineered. Voltage clamp and 20 Hz 400/470 nm imaging detected 400 µV steps. CRISPR made Cx43 and Cx43/Cx45 knockouts. Correlation, Ca2+ imaging and cluster tests probed coupling.

Results: rEstus2s resolved 0 to -100 mV with 4.6-fold change. ANO1/KCa3.1 drove isolated-cell fluctuations. Gap junctions suppressed them at confluence (BCI). Double knockout abolished suppression; Cx43 rescue restored it. Vm variance scaled ~1/n.
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