Cholesterol Depletion with (2-Hydroxypropyl)- β-Cyclodextrin Modifies the Gating of Membrane Electroporation-Induced Inward Current in Pituitary Tumor GH3 Cells: Experimental and Analytical Studies

摘要

The effects of (2-hydroxypropyl)- β-cyclodextrin (HPβCD), a cyclic oligomer, on membrane electroporation-induced inward current (iI/isubMEP/sub) in pituitary tumor (GHsub3/sub) cells were experimentally and analytically characterized. Depletion of membrane cholesterol by exposing cells to HPβCD (2 mM) increased the activation time constant of delayed rectifier Ksup+/sup current. Such maneuver resulted in a significant reduction of iI/isubMEP/sub density. 2,2’-Azo-bis(2-amidinopropane) dihydrochloride (AAPH), an initiator of free radicals, increased the magnitude of iI/isubMEP/sub. AAPH-stimulated iI/isubMEP/sub was not reversed by the blockers of Casup2+/sup-activated Ksup+/sup channels, but by LaClsub3/sub or MnClsub2/sub. However, in HPβCD-treated cells, the ability of AAPH to enhance iI/isubMEP/sub was abolished. Under such maneuver, the gating charge of iI/isubMEP/sub activation was increased by 2 fold, along with a hyperpolarized shift of the activation curve by 30 mV. No change in single-channel conductance of MEP-induced channels during cell exposure to HPβCD was demonstrated. The energy change of iI/isubMEP/sub in untreated and HPβCD-treated cells was estimated to be -17.7 and -44.8 kJ/mol, respectively, and the perturbation of free energy following HPβCD treatment was -27.1 kJ/mol. Based on an MEP model, cell exposure to HPβCD increased the edge energy of the electropore size. By use of a two barrier-one site barrier model, HPβCD treatment can increase both the peak height and well depth of the barrier profile. Taken together, depletion of membrane cholesterol by HPβCD can elevate the edge energy of pore formation, thereby decreasing the iI/isubMEP/sub magnitude. The channel-suppressing properties during membrane cholesterol depletion with HPβCD might thus contribute to the underlying mechanisms by which such maneuver alters neuronal or neuroendocrine function.