The oocyte was placed in a recording chamber with bath volume of 500 ��l and impaled with two electrodes. Micropipettes were made from VWR calibrated (100 ��l) borosilicate glass capillaries (World Precision Instruments) selleck catalog pulled with a Kopf model 700D vertical pipette puller (Kopf Instruments) and had resistances of 0.5�C2.0 M�� when filled with 3 M KCl and inserted into the bath solution. A SF-77B Perfusion Fast-Step (Warner Instruments, Hamden, CT) set at 3 steps/position was used for rapid exchange of solution bathing the oocyte from ND96 pH 7.4 to ND96 pH 4.0 (with 5 mM MES substituted for 5 mM HEPES) to activate the heterologously expressed hASIC1b channel. The protocol used exposed the oocyte to pH 7.4 solution for 13 s, to pH 4.0 for 13 s, and again to pH 7.

4 for 13 s to allow for complete recovery of the acid-activated channel. Before acid-activated currents were recorded, the clamp gain and stability were set for each oocyte using capacitive transients of a step protocol from ?40 to ?50 mV. Oocytes were clamped at ?60 mV for all the experiments. The flow of solutions out of the perfusion fast step was controlled with a VC-6 Six Valve Controller (Warner Instruments), and the solution flow rate was 1�C2 ml/min. For pH activation curves, the solution flowing out of one barrel of the perfusion system was ND96 pH 7.4 while the solution flowing out of the second barrel was changed to ND96 pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.0 sequentially. These solutions were buffered with 5 mM HEPES (for pH 7.4 to 6.0) or 5 mM MES (for pH 5.5 to 4.0).

Acid-activated currents at each pH were normalized to the peak pH 4.0 current (IpH4.0) of the oocyte. Pooled normalized values were fitted to the sigmoidal dose-response (variable slope) equation in GraphPad Prism 3 to obtain pH50 values and Hill coefficients. For PKC activator experiments, five sweeps of acid-activated currents (activated with ND96 pH 4.0 solution) were recorded, the flow of solutions was stopped, and the oocyte was unclamped before and during the addition of PMA or PdBu to the bath solution to a final 1 ��M concentration for 5 min (21). After the PMA incubation period, the oocyte was clamped at ?60 mV, and another five acid-activated Dacomitinib peaks were recorded. As controls, we used 5 min of no treatment, DMSO (1:1,000, the equivalent of DMSO in the PMA experiment), or 4-��-PMA (an inactive PMA analog) at a final concentration of 1 ��M. The peak acid-induced current recorded after the addition of a treatment (no treatment, DMSO, 4-��-PMA, PMA, or PdBu) was normalized to the current from the last sweep before treatment. In some experiments, oocytes were preincubated in ND96 pH 7.4 with 1 ��M chelerythrine for 1 h before recording.