Structural determinant of TRPV1 desensitization interacts with calmodulin

Abstract

The capsaicin receptor, TRPV1 (VR1), is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. Extracellular Ca2+-dependent desensitization of TRPV1 observed in patch-clamp experiments when using both heterologous expression systems and native sensory ganglia is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. Here, we show that the Ca2+-binding protein calmodulin binds to a 35-aa segment in the C terminus of TRPV1, and that disruption of the calmodulin-binding segment prevents TRPV1 desensitization. Compounds that interfere with the 35-aa segment could therefore prove useful in the treatment of pain.

Fig. 2.

In vivo and in vitro interaction of calmodulin with TRPV1. (A) TRPV1 immunoprecipitation analysis in HEK293 cells transfected with Myc-tagged CaM and TRPV1 or pcDNA3 with or without capsaicin (Cap) treatment (1 μM, 3 min) in the presence of Ca²⁺. CaM was coimmunoprecipitated with TRPV1 depending on Cap treatment. Similar CaM expression was confirmed in both pcDNA3- and TRPV1-transfected cells regardless of Cap treatment. (B) Analysis of CaM binding to cytoplasmic domains of TRPV1 by using a GST pull-down assay in the presence of Ca²⁺. Only the C terminus of TRPV1 was found to interact with CaM. (C) Identification of a CaM-binding segment in the C terminus of TRPV1 by using a GST pull-down assay in the presence of Ca²⁺. Four C-terminal segments of TRPV1 used for making GST-fusion proteins are shown in the context of a putative transmembrane topology model (Left). (Right) A pull-down assay using GST-fusion proteins with four segments. (D) GST pull-down assays with C terminus of TRPV1 (Left), C terminus lacking 35 aa (Left), and segment 2 of C terminus (Right) in the presence (+) or absence (-) of Ca²⁺.

Fig. 1.

Extracellular Ca²⁺ dependence of capsaicin receptor current desensitization. (A and B) Representative traces of capsaicin-evoked whole-cell currents by short, repetitive application in HEK293 cells expressing WT TRPV1 in the absence (A) or presence (B) of extracellular Ca²⁺. Cells were held at -60 mV. (C) Representative trace of WT TRPV1 current evoked by capsaicin with CaM inhibitor W7 (100 μM) in the pipette solution in the presence of extracellular Ca²⁺. (D) Representative trace of capsaicin-evoked current in cells expressing WT TRPV1 and mutated CaM (D21A/D57A/D94A/D130A) in the presence of extracellular Ca²⁺.

Fig. 3.

CaM-binding segment of TRPV1 is sufficient for the extracellular Ca²⁺-dependent desensitization. (A) Representative trace of capsaicin-evoked current by short, repetitive application in HEK293 cells expressing deletion mutant of TRPV1 (Δ35AA) in the presence of extracellular Ca²⁺.(B) Representative traces of current responses evoked by long application of capsaicin in cells expressing WT TRPV1 (Left and Center)or Δ35AA (Right) in the absence (Left) or presence (Center and Right) of Ca²⁺. (C and D) Extent of desensitization summarized from the data in the short application (C) and the long application (D) protocol. The current ratios (mean ± SE) of the second to the first of two consecutive, 10-sec applications with a 30-sec interval (C) or the residual fractions (mean ± SE) of peak currents remaining at application of 1 μM capsaicin for 30 sec (D) are shown. *, P < 0.01 vs. WT TRPV1 with Ca²⁺; **, P < 0.01 vs. Δ35AA, two-tailed unpaired t test. (E) Effects of deleting a CaM-binding domain on desensitization of the TRPV1 currents induced by short, repetitive acid stimulations in the presence of extracellular Ca²⁺. The ratios (mean ± SE) of the second to the first of two consecutive, 10-sec applications of acidic solution (pH 4.0) are shown. *, P < 0.01 vs. WT TRPV1 with Ca²⁺; /#, P < 0.05 vs. WT TRPV1 with Ca²⁺, two-tailed unpaired t test.