Figure 5

Potentiation of ASIC1a/2a by low concentration PcTx1 in mouse cortical neurons. (a) Correlation between pH6.8-induced current and membrane depolarization in wild-type (solid squares; n = 17) and ASIC1^−/− (open circles; n = 16) neurons. Each symbol represents one neuron. (b) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of 10 nM PcTx1. Inset: Time course of the effect of 10 nM PcTx1 and wash. The dotted line at the bottom represents the baseline. (c) Time courses of pH6.8-induced responses upon removal of 10 nM PcTx1. Wash began at t = 0 sec as indicated by the green arrow. Open squares: current (n = 4); solid squares: membrane potential (n = 4). Responses were normalized to the control values for each cell just before application of 10 nM PcTx1. Note the initial lower responses (at t = 60 sec) than those in the presence of 10 nM PcTx1 (at t ≤ 0 sec). (d) Summary of pH6.8-induced membrane depolarization in wild-type and ASIC1^−/− neurons. Values for wild-type neurons during and upon initial wash of 10 nM PcTx1 were both significantly higher than that from ASIC1^−/− neurons (p < 0.05 and p < 0.0001, respectively). Inset: pair-wise comparison of pH6.8-induced membrane depolarization in the presence of 10 nM PcTx1 and 1 min after wash-off. The former was larger than the latter for all the four wild-type neurons tested, despite not reaching statistical significance (p = 0.11). A typical protocol used for experiments in Fig. 5 was as follows. For current recording, the holding potential was −80 mV; for membrane potential recording, a small hyperpolarizing current (0–100 pA) was injected to maintain the resting membrane potential near −70 mV. The conditioning pH was 7.4 and the test pH of 6.8 was applied for 2 sec before returning to pH7.4. This pH application protocol was repeated once every 60 sec. Where applicable, PcTx1 was present in both conditioning and test pH buffers for 4–8 min till reaching steady state before wash.