FIGURE 2.
Effects of CDs on KV1.3 current.
(A,B) Patch-clamp measurements were performed in outside-out or whole-cell configurations in CHO cells transiently expressing KV1.3. Two representative measurements with the application of 1mM (panel A) and 5mM (panel B) iαCD are shown. K+ currents were elicited by applying 15ms-long depolarizing pulses from −100mV to +50mV every 15s (panels A and B, top). First, cells were perfused with standard extracellular solution to obtain control currents (I 0 , panels A and B, black solid lines). To validate proper working of the perfusion system an extracellular solution with 150mM K+ was applied for each cell prior to CD exposures (panel A, grey line). Cells were perfused with CD-containing extracellular solutions for a maximal application time of 3min to avoid cholesterol-extracting effects of CDs (panels A and B blue lines). Then the reversibility of current-blocking effects induced by the compounds was determined by the reapplication of standard extracellular solution (panel A and B black dotted lines). (C,D) To demonstrate the wash-in kinetics of CDs, leak-corrected peak currents at every time point were normalized to the maximal peak. Among cholesterol-depleting CDs, only MβCD in 5mM exhibited a current block (Panel C, black dashed line), while among the non-cholesterol depleting inverted CDs, iαCD (panel D, blue lines) and iγCD (panel D, cyan lines) showed similar effects at both 1mM (solid lines) and 5mM (dashed lines) concentrations. The current blocking effects were saturated within 90s after the initiation of CD exposure in all cases. (E) To quantify current-inhibiting effects, remaining current fraction (RCF) values were calculated according to equation (Davis and Brewster, 2004). The efficacy order of the induced current block was MβCD < iαCD < iγCD in both concentrations. (F) To characterize the extent of wash-out, recovered current fractions (RF) were determined according to equation (Uekama, 2004). This analysis showed that the current block induced by CDs was only partially reversible. Data represented as mean ± SEM based on n = 4–6 cells.