Gap Junction Channels and Hemichannels by Donglin Bai and Juan C. Sáez

Author:Donglin Bai and Juan C. Sáez
Language: eng
Format: epub
Publisher: CRC Press

5.5.7.4 Analysis

1. The fluorescent values from both cells, obtained by subtracting a noncellular area (background) from equivalent brightest areas on each cell, are plotted as a function of time. The recipient values are fit with a numerical method (stated immediately below (in the next paragraph) and illustrated in Figure 5.7) to calculate the diffusion rate constant (k) between the donor and the recipient.

2. The rate constants (k) are obtained by fitting each time (t) point with the equation: C2(t + Δt) = C2(t) + kΔt/2[C1(t) + C1(t + Δt) - C2(t) – C2(t + Δt)], where At is the time between points, C1 is the donor, and C2 is the recipient fluorescence (cf. Ek-Vitorin and Burt 2005; Ek-Vitorin et al. 2006).

3. Using the solver feature in Excel (Figure 5.7), k is varied to minimize the RMS between the predicted and experimental values of C2(t). k from each junction is plotted as a function of the corresponding electrical conductance value, calculated as gj = Ij/Vj.

4. The analysis of the transjunctional dye coupling with the whole cell patch clamp style injection is based on the assumptions that (1) the diffusion occurs between two equal compartments and is contingent upon the coupling level and the instantaneous dye gradient between them; (2) the pipette is a virtually inexhaustible source of dye for the duration of the recording; (3) the donor cell dye concentration plateaus toward that of the pipette, followed by the recipient with a delay dependent on the coupling level; (4) no significant changes in the channel function occur during the entire recording period, unless induced by the researcher (Ek-Vitorin and Burt 2005).

5. If permselectivity were constant, a plot of k versus gj for multiple junctions would describe a linear relationship that represents the signature of a connexin type. At least for Cx43 that has not been the case, indicating that channels in different functional states (some more and some less permeable to the dyes compared to current-carrying ions) exist in the junction (Ek-Vitorin et al. 2006).

Download

Copyright Disclaimer:
This site does not store any files on its server. We only index and link to content provided by other sites. Please contact the content providers to delete copyright contents if any and email us, we'll remove relevant links or contents immediately.