Fig. 2.

Capsaicin opens the TRPV1 pore to facilitate cellular entry of YO-PRO-1. (A) 1 μM capsaicin induced YO-PRO-1 uptake in rat TRPV1-expressing cells. (B) The fluorescent signals were quantified with plate-reader assays. TRPV1-dependent fluorescent signals, indicating YO-PRO-1 entry, were concentration dependent. (C) Application of the TRPV1 pore blocker ruthenium red (RR, 10 μM) suppressed capsaicin-mediated YO-PRO-1 uptake (Upper, gray trace). To test the time dependence of ruthenium red block, cells were preincubated in capsaicin for 2 h and then the agonist was removed before the subsequent YO-PRO-1 uptake experiments. Ruthenium red (10 μM), when applied concurrently with YO-PRO-1, quickly and effectively blocked the uptake (comparing the two black traces in the chart). (D) Pretreatment with 200 μM Ba²⁺ also blocked capsaicin-induced YO-PRO-1 uptake (72.1 ± 0.7% block, n = 3 wells). Ba²⁺ effects could be completely reversed (94.7 ± 3.4% recovery, n = 3 wells) by coapplying 1 mM EGTA, a chelator for this divalent cation, with YO-PRO-1. (E) 5 μM YO-PRO-1 reversibly blocked TRPV1 currents evoked by 10 μM capsaicin in an inside-out patch. The i-V plot shows a weak voltage dependence of YO-PRO-1 block.