Functional contribution of DPPX to transient outward K currents of rabbit CB chemoreceptor cells

(A) Validation of siRNA DPPX was performed, quantifying the amount of DPPX protein by Western blot and the amount of DPPX mRNA by real-time PCR. The figure shows a Western blot of lysates from HEK cells untransfected (control lane) or transfected with DPPX alone (DPPX) or DPPX+DPPX siRNA (siRNA lane), probed with anti-DPPX antibody. The arrow indicates the estimated molecular weight of DPPX protein. The reduction of the levels of DPPX mRNA was determined by qRT-PCR and analyzed with the 2 method as described in the Materials and methods section. Total mRNA was extracted from HEK cells transfected with rabbit DPPX alone or in combination with DPPX siRNA as indicated in the figure. (B) Currents recorded from two individual CB chemoreceptor cells after electroporation of GFP + negative control siRNA (siRNA nC) or GFP+DPPX siRNA (siRNA DPPX). In both cases, the currents were elicited with the voltage protocol shown at the bottom, in which 500-ms depolarizing steps to +40 mV follow 6.5-s prepulses to two different potentials, −80 mV (thicker trace) and 0 mV (thinner trace). The difference between the current amplitude at +40 mV in these two pulses is defined throughout the paper as the transient outward current. The black thick line shows the fit of the currents to a double-exponential function. (C) Inactivation fitting parameters obtained in both conditions (control and siRNA cells) indicate a significant change in the relative amplitudes of the two components of inactivation, A and A. The bar plot shows the relative proportion of the fast component. Data are mean ± SEM of 8–10 cells in each group; *, P < 0.05. (D) Peak current densities recorded in control or siRNA DPPX electroporated cells were obtained with the two pulse protocol described in B. I represents the peak current amplitude at +40 mV after the −80-mV prepulse, I was calculated as indicated above, and I is the current amplitude at +40 mV after the 0-mV prepulse. *, P < 0.05, = 10 cells in each group. (E) The normalized steady-state inactivation curves and the conductance–voltage curves from the two groups of cells were obtained and constructed as described in . Lines represent the best fit of the data to Boltzmann functions. Each point is the mean ± SEM of 8–10 determinations. The bar plot shows the differences in the V for activation obtained from the individual fit of the cells in both groups.<p><b>Copyright information:</b></p><p>Taken from "A Role for DPPX Modulating External TEA Sensitivity of Kv4 Channels"</p><p></p><p>The Journal of General Physiology 2008;131(5):455-471.</p><p>Published online Jan 2008</p><p>PMCID:PMC2346566.</p><p></p