Figure 1.

(A–B) Direct transfection of siRNA Cy-3 targeting mHCN2 in single cells. (A) Properties of mHCN2 channels. Voltage protocol (upper panel) and whole-cell currents (I_m) recorded in untransfected (black current traces) and mHCN2-transfected (green current trace) HEK293 cells. Red current traces represent mHCN2-transfected cells that are also transfected with siRNA (siRNA Cy-3) against mHCN2. (B) Average current densities measured at −140 mV from HEK293/HCN2 control cells (green bar: 62.1± 6.7 pA/pF, n = 28) and siRNA-transfected cells (pink bar: 1.5 ± 0.5 pA/pF, n = 10), P<0.001. (C) Detection of cell-to-cell transfer of siRNA in isolated cell pairs. Fluorescent intensity over time for source cell (•) and recipient cell (○). Upper insert: bright field image (left) and fluorescent image in pseudo-colors (right) of a HeLaCx43 cell pair where a patch electrode containing siRNA Cy-3 is in whole-cell configuration with one of the two cells (source cell). The fluorescent image was taken 11 min after establishing a whole-cell patch in the source cell. Fluorescence can be seen in the recipient cell. To enhance visibility of siRNA Cy-3 in the recipient cell, fluorescence is shown here in pseudo-color scale from the lowest intensity (blue, background) to red (highest intensity). After 11 min a second electrode was used to patch the recipient cell and junctional conductance of g_j = 35 nS was determined). Scale bar, 20 μm Lower insert: expanded scale for recipient cell.