Figure 6. Native MiRP2 slows and downregulates native delayed rectifier currents in PC12 cells. A, RT-PCR of Kv2.1 (top) and Kv3.1 (bottom) from mRNA isolated from PC12 cells. Lanes indicate amplification by PCR from: lane 1, water control; lane 2, mRNA from PC12 cells after RT-PCR; lane 3, mRNA from PC12 cells before RT-PCR; lane 4, plasmid DNA containing Kv2.1 (top) or Kv3.1 (bottom); lane 5, marker. Expected product sizes for gene fragments, indicated by labeled lines, were 557 bp for Kv2.1 and 522 bp for Kv3.1. B, RNAi gene silencing of endogenous MiRP2 assessed by semiquantitative PCR of products from RT-PCR of PC12 cell mRNA. Top gel, Normalization of RT-PCR products by titration of amplified β-actin; lanes show approximately equal β-actin band intensities. Bottom gel, RT-PCR for rMiRP2 using normalized samples. Top and bottom gels, Lane 1, Marker; lane 2, water control; lane 3, nontransfected PC12 cell RT-PCR product; lane 4, MiRP2 siRNA-transfected PC12 cell RT-PCR product; lane 5, MiRP2 cDNA-transfected PC12 cell RT-PCR product. Expected product sizes for gene fragments, indicated by labeled lines, were 450 bp for actin, and 596 bp for MiRP2. C, Exemplar traces showing potassium currents recorded using protocol 4 (inset) in nontransfected PC12 cells (black circle, untrans) or PC12 cells transfected with MiRP2 cDNA (open circle, M2 cDNA), MiRP2 siRNA (gray square, M2 siRNA), or scrambled control siRNA (black square, con siRNA). Cotransfection with blank plasmid was used to keep total transfected nucleic acid concentration equal. D, Mean peak current for nontransfected PC12 cells (black circle; n = 39) or PC12 cells transfected with MiRP2 cDNA (open circle; n = 16), MiRP2 siRNA (gray square; n = 14), or scrambled control siRNA (black square; n = 16); protocol as in C. Error bars indicate SEM. The double asterisks indicate significant differences between current densities of MiRP2 cDNA transfected versus MiRP2 siRNA or nontransfected cells at all positive voltages (Mann-Whitney rank sum test; p < 0.001). The single asterisk indicates significant difference between current densities of MiRP2 siRNA transfected versus nontransfected cells at voltages between +30 and +60 mV (Mann-Whitney rank sum test; p < 0.05). E, Mean inactivation of PC12 cell outward potassium currents at +60 mV expressed as percentage inactivation over 1 sec, in nontransfected (untrans) and transfected PC12 cells as indicated, calculated from the same traces as in D. The double asterisk indicates significant difference between mean inactivation of MiRP2 cDNA transfected versus MiRP2 siRNA-transfected cells (unpaired t test; p < 0.001). F, Trace resulting from subtraction of averaged +60 mV trace for MiRP2 cDNA-transfected PC12 cells from averaged +60 mV trace for MiRP2 siRNA-transfected PC12 cells; protocol (inset) as in C. G, Mean activation rates of nontransfected (untrans) and transfected PC12 cells as indicated, calculated from the same recordings as in D. Activation traces at 0 mV were fitted with a double exponential function yielding fast and slow τact components. Error bars indicate SEM. A single asterisk indicates a significant difference between the slow components of τact of PC12 cells transfected with control siRNA and those transfected with MiRP2 siRNA (unpaired t test; p < 0.05). Relative amplitudes of the slow components of activation were not significantly different and were 0.63 ± 0.05 (nontransfected), 0.54 ± 0.1 (MiRP2 cDNA), 0.58 ± 0.08 (MiRP2 siRNA), and 0.55 ± 0.1 (scrambled control siRNA). H, Mean deactivation rates of nontransfected (untrans) and transfected PC12 cells as indicated, calculated from the same recordings as in D. Deactivation traces at -30 mV were fitted with a double exponential function yielding fast and slow τdeact components. Error bars indicate SEM. Relative amplitudes of the slow components of deactivation were not significantly different and were 0.31 ± 0.03 (nontransfected), 0.25 ± 0.09 (MiRP2 cDNA), 0.25 ± 0.09 (MiRP2 siRNA), and 0.38 ± 0.06 (scrambled control siRNA).