Figure 2
Development of Best1 specific genetic tools. A) Left; Topology model of Bestrophin suggested by Tsunenari et al [17]. Blue circle indicates amino acid position of Best1 pore mutant (W93C). Red square indicates target amino acids for Best1 shRNA. Right; nucleotide and amino acid sequences of Best1 shRNA target. Nucleotide sequence differences from wild type Best1 (Best1 WT) and shRNA insensitive Best1 (Best1(sh insens)) were highlightened. B) Schematic diagram for perforated-patch-current recording from cultured astrocytes. C) Representative perforated-patch-current recording from Best1-shRNA and Best1 (sh insens) or Best1-shRNA and shRNA insensitive pore mutant Best1(Best1-W93C (sh insens)) expressing cultured astrocytes. The current responses were recorded in response to a voltage ramp command(from -100 to +100 mV, 1 s duration, 0.2 Hz; Vh of -70 mV) before and after 30 μM TFLLR treatment. D) The bar graph summarizes the mean current amplitude at Vh = -70 mV ± s.e.m (*p < 0.05, **p < 0.01, unpaired t-test). Among 13 recorded astrocytes which expressed Best1-shRNA and Best1 (sh insens), only 5 cells showed comparable currents. And a big current recorded in an astrocyte expressing Best1-shRNA and Best1-W93C (sh insens) was eliminated in the bar graph. Numbers of determinations are indicated on the bar graph.