Figure 2.

Alkylation using MTSEA leads to a suppression of the electrogenic response in oocytes expressing mutant S460C. (A) Comparison of the P_i-induced current for an oocyte expressing the WT NaPi-IIa before and after exposure to 100 μM MTSEA (left) and two oocytes expressing S460C before and after exposure to 100 μM MTSEA or MTSET for 2 min (right). Bars represent time of application of 1 mM P_i. Dashed line indicates baseline holding current level. Note that P_i induces an upward deflection in the holding current after alkylation. (B) Restoration of P_i response by 15-min incubation in 10 mM DTT (right) for a cell expressing S460C. Incubation in 1.5 μM MTSEA exposure (middle) inhibited the initial P_i response (left) by 80%. (C) Dependency of the P_i response on MTSEA concentration. Inset gives a set of original records showing P_i response (applied during bar) for an oocyte expressing S460C after exposure to successive 10-fold increasing doses of MTSEA from 0.001 to 10 μM. Scale: vertical 50 nA, horizontal 20 s. For this cell, after exposure at 10 μM MTSEA, P_i induced an upward deflection of the baseline current. There was no change in the response after exposure to 100 μM MTSEA. Dose response is pooled from five oocytes. The P_i-induced response was calculated relative to the P_i-induced current after alkylation in 100 μM MTSEA, and was then normalized to the initial response for each cell. Curve is a fit of to the data, which gave an apparent half-maximal concentration = 0.5 μM.