2D). As shown in Fig. 2E, single-channel currents evoked by CAP exhibited outward rectification, and the single-channel slope conductance was 45.6 �� 2.1 pS at �C60 mV and 72.1 �� 10.8 pS at 60 mV, consistent with previous reports (Oh?et al.?1996; Jung?et al.?1999;?n?= 5). To provide more detailed evidence that intracellular acidification attenuated?iCAP, we examined the effects of an acidic http://www.selleckchem.com/
bath solution using isolated membrane patches in inside-out configuration. Openings of single channels at �C40 mV in symmetrical 143 mm K+ are shown in Fig. 3A. Capsaicin (300 nm) activated?iCAP rapidly. When an acidic (pH 6.4) bath solution containing 300 nm CAP was applied to the same patch, channel activity decreased significantly (Fig. 3A). These results are summarized in Fig. 3B, which shows that the channel activity of?iCAP, expressed as?NPo, was significantly attenuated by intracellular acidification (0.69 �� 0.2 at pH 7.4; 0.40 �� 0.1 at pH 6.4;?P < 0.05;?n?= 5). Amplitude histograms were obtained from channel openings at �C40 mV (Fig. 3C). Single-channel slope conductance at �C40 mV was not affected by Tryptophan synthase
intracellular acidification (50.3 �� 4.6 pS at pH 7.4 and 51.0 �� 3.5 pS at pH 6.4;?n?= 5; Fig. 3D). These data suggest that intracellular acidification attenuates TRPV1 channel activity in rat DRG neurons not by decreasing the amplitude of single-channel conductance, but by decreasing the probability of an open?iCAP. To determine whether intracellular acidification caused by a decrease in pHo affected the activity of TRPV1, we measured?ICAP in cell-attached patches to protect TRPV1 channels located in the membrane patches from potentiating in the acidic bath solution. In this configuration, capsaicin and capsazepine applied in the buy Decitabine
bath solution can readily activate and block TRPV1 because they are lipid soluble and can cross the membrane to access the channels. In addition, a low pHo in the bath solution produced intracellular acidosis exclusively without affecting TRPV1 activity. As shown in Fig. 4A, when CAP (300 nm) was added to the usual (pH 7.4) bath solution, a rapid inward current developed, similar to Fig. 2A. This current was significantly decreased in a reversible manner by application of the acidic (pH 6.4) bath solution containing CAP (300 nm) and abolished by CZP (10 ��m; Fig. 4A?and?B). These results are summarized in Fig. 4C and suggest that the channel activity of?iCAP, expressed as?NPo, was significantly attenuated by intracellular acidification induced by a decrease in pHo (2.09 �� 1.4 at pH 7.4, 72.0 �� 7.4 at pH 7.4 with or without CAP, 35.71 �� 7.8 at pH 6.4 with or without CAP, 5.53 �� 1.6 at pH 7.4 with or without CAP with or without CZP; pH 7.4 with CAP?versus?pH 6.4 with CAP,?P < 0.05;?n?= 5). Extracellular acidosis readily potentiates TRPV1 channel activity (Tominaga?et al.?1998; Baumann & Martenson, 2000; Neelands?et al.?2005), while intracellular acidosis attenuates TRPV1 channel activity, as shown in Figs 1 and 3.