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. 2015 Apr 8;6:63. doi: 10.3389/fphar.2015.00063

Figure 6.

Figure 6

PI(4,5)P2 depletion does not alter voltage dependence of VSD motions. (A) Current trace showing the activation of the endogenous Ca2+-activated chloride channel following addition of serotonin in oocytes expressing the 5HT2C receptor and Ci-VSP. 5HT2C receptor activation of the oocyte PLC pathway results in the conversion of PI(4,5)P2 into IP3 and DAG. The IP3 causes an increase in intracellular Ca2+ leading to the activation of the chloride channel. hp = −80 mV. (B,C) TMRM FVs before (black) and 10 min after (magenta) application of 10 μM serotonin. Activation of PLC through the 5HT2C receptor leads to depletion of PI(4,5)P2 in the membrane and results in a shift in the DA* (B) and DA/quadK* (C) TMRM FVs after addition of serotonin. In both cases, the VSD motions are sensitive to the PI(4,5)P2 in the membrane, indicating that the DA/quadK* may not depend on PI(4,5)P2 in the membrane to cause the activity shifts seen in Figure 4. DA* (no serotonin) V1/2 = 21.8 ± 0.3, slope = 19.9 ± 0.2; DA* (serotonin) V1/2 = 31.7 ± 0.6, slope = 26.2 ± 0.5; DA/quadK* (no serotonin) V1/2 = 48.3 ± 0.2, slope = 20.7 ± 0.2; DA/quadK* (serotonin) V1/2 = 54.0 ± 0.6, slope = 25.0 ± 0.5. All error bars are ± s.e.m. (D) Representative TMRM fluorescence traces during a step from an hp of −80 to +200 mV for WT* (black), DA*(gray), and DA/quadK* (purple). Traces are normalized to the maximal fluorescence change. DA* significantly slows down the repolarization kinetics while DA/quadK* kinetics were faster. (E) Representative TMRM fluorescence traces during a step from an hp of −80 to +200 mV for DA* (black), DA/K553Q* (rust), DA/K554Q* (orange), DA/K555Q* (blue), and DA/K558Q* (green). Traces are normalized to the maximal fluorescence change. The voltage trace reports the actual voltage recorded during acquisition. Inset shows full acquisition with the fluorescence returning to baseline. Scale bar for inset is 100 ms. All Lys to Gln mutations in the D331A background maintained the slow or slower kinetics from DA* alone. The individual mutations do not sum to equal the effect observed for DA/quadK* in (D).