Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4) channels/mu m(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C-m). The C-m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C-m of 1.15 +/- 0.08 mu F/cm(2) in ChR2-expressing; cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (039 +/- 0.02 mu F/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C-m (0.85 +/- 0.07 mu F/cm(2)). as compared to non-expressing control cells (0.70 +/- 0.03 mu F/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins. (C) 2008 Elsevier Inc. All rights reserved.