Phosphoinositides such as PIP2 are emerging as important modulators of TRP channel activity. Here, we demonstrate that Pirt, a membrane protein, binds both PIP2 and TRPV1 and positively regulates TRPV1 via PIP2. Pirt is highly conserved among vertebrates, and no closely related invertebrate homologs were found by a genome-wide sequence search, suggesting that vertebrates have acquired additional regulatory subunits for TRP channels. The expression of Pirt is restricted to the PNS (predominantly DRG and trigeminal ganglia) and is not found in the central nervous system (CNS). Therefore, the modulation of TRPV1 activity by Pirt likely occurs in these peripheral neurons but not in higher-order neurons within the CNS. Since Pirt is also expressed in TRPV1-negative neurons in DRG, it may be involved in regulating other TRP channel activity.
Pirt−/− mice show noxious heat and capsaicin behavioral phenotypes that resemble, but are less severe than, those observed in TRPV1−/− mice. These data support the hypothesis that TRPV1 works as a primary sensor of noxious heat and capsaicin, while Pirt regulates TRPV1 activity. Pirt itself does not exhibit any channel activity upon noxious heat or capsaicin treatment (data not shown). Whole-cell recordings from DRG neurons and TRPV1 stably expressing HEK293 cells suggest that Pirt enhances TRPV1-mediated currents. This enhancement is not due to altered cell surface level expression of TRPV1 by Pirt based on our surface biotinylation data. Single-channel recording experiments should give us more detailed information on how Pirt affects conductance, open time, or open probability of TRPV1.
Consistent with a role for Pirt in potentiating TRPV1 sensation, we found that Pirt physically interacts with TRPV1. Therefore, it is likely that Pirt functions as a regulatory subunit of the TRPV1 complex. More importantly, the C terminus of Pirt alone can bind PIPs and TRPV1 and enhance heat-evoked current through TRPV1. The basic residues in the C terminus of Pirt are required for PIP2
but not TRPV1 binding, suggesting that the 26 amino acids of the C terminus contain separate binding domains for PIP2
and TRPV1. In addition, the mutant form of the Pirt C terminus failed to modulate TRPV1 function, indicating that PIP2
binding is essential for Pirt regulation of TRPV1 and that the binding to TRPV1 alone is not sufficient to enhance the channel's activity. The C terminus of Pirt can bind to PIPs other than PIP2
in vitro, suggesting that nonspecific electrostatic interactions contribute strongly to the binding between the basic residues and the negatively charged inositides. Such interactions with PIPs are well characterized for several proteins, such as MARCKS, which contain clusters of positively charged residues (Wang et al., 2001
). The broad PIP binding of Pirt is consistent with other findings that TRPV1 can be modulated by various PIPs besides PIP2
(Kwon et al., 2007
; Lukacs et al., 2007
). Therefore, it is probable that other PIPs may also participate in Pirt's regulation of TRPV1.
It has been shown that PIP2
has an inhibitory effect on heat- and capsaicin-evoked TRPV1 current (Chuang et al., 2001
; Prescott and Julius, 2003
). However, other studies have found that PIP2
has an activating effect on TRPV1. For example, direct application of PIP2
to the inner leaflet of TRPV1-expressing membranes led to channel activation, while sequestering PIP2
with polylysine inhibited channel function (Stein et al., 2006
). A more recent study may reconcile the discrepancy (Lukacs et al., 2007
). These authors found that PIP2
has a dual effect on TRPV1: inhibitory at low capsaicin concentrations and activating at high capsaicin concentrations. Our data support previous findings that PIP2
can have both activating and inhibitory effects on TRPV1 and highlight the role of Pirt in mediating the activating effect of PIP2
application experiments suggest that PIP2
has an activating effect on TRPV1 at high capsaicin concentrations (5 μM). Importantly, Pirt is required for this positive effect. Other groups have observed PIP2
activating effects on TRPV1 activity in heterologous systems where Pirt is unlikely to be present (Stein et al., 2006
; Lukacs et al., 2007
). In these cases, the experiments were performed using inside-out recording with direct application of PIP2
to the inner leaflets of membranes. Therefore, it is likely that the very high levels of PIP2
applied in their experimental systems can bypass the requirement for Pirt. Furthermore, in our capsaicin dose-response experiments, the activating effect of Pirt on TRPV1-mediated currents was only prominent when high concentrations of capsaicin (5 and 10 μM) were applied. This effect is also PIP2
dependent (). Therefore, both Pirt and PIPs are required for optimal TRPV1 function.
At low concentrations of capsaicin, PIP2
plays an inhibitory role on TRPV1. It has been proposed that the inhibitory effect maybe indirect and that other PIP2
-binding protein(s) may be involved (Stein et al., 2006
; Lukacs et al., 2007
). Our data show that Pirt is not involved in this modulation since Pirt can further enhance the activity of TRPV1Δ42. Therefore, Pirt plays an essential role only in the activating effect of PIP2
but not in its inhibitory effect. These results also indicate that the inhibitory effect of PIP2
at low capsaicin concentration masks the activating effect of Pirt on TRPV1. However, removal of the inhibitory effect by bradykinin-activated PLC or deleting the last 42 amino acids of TRPV1 unmask the activating effect of Pirt. We have shown that PIP2
modulates TRPV1 in two ways: inhibition by interacting with the last 42 amino acids of TRPV1 and activation via association with Pirt at a different region of TRPV1. Furthermore, hydro-lysis of PIP2
by PLC may differ in these situations.
Our behavioral and cellular studies show that Pirt does not play a role in TRPA1 function, suggesting that Pirt does have specificity toward certain TRP channels. Interestingly, Pirt also fails to bind TRPA1. It will be interesting to test if the failure of Pirt to regulate TRPA1 is due to its inability to bind this channel. However, we cannot rule out the possibility that Pirt affects the activities of other TRPs, especially as PIP2
has been implicated in the regulation of many TRP channels (Hardie, 2007
; Voets and Nilius, 2007
). For example, TRPM8, the cold and menthol sensor (Colburn et al., 2007
; Dhaka et al., 2007
), requires PIP2
for activation (Rohacs et al., 2005
). Interestingly, Pirt can also bind TRPM8. Therefore, Pirt may regulate the activities of other TRP channels via PIP2
. Because TRP channels play critical roles in nociception, uncovering the interactions between Pirt, TRPs, and PIPs should provide a better understanding of pain transduction.
The arms of the Pirt
targeting constructs were subcloned from a 129/SvJ genomic DNA lambda phage clone (Invitrogen) and ligated with an EGFPf-IRES-rtTA-CAN cassette. Pirt+/−
mice were generated using the targeting construct by a transgenic mouse facility at the University of Cincinnati. Details of the generation of Pirt
knockout mice are available in the Supplemental Data
. The DNA fragments corresponding to the N- (1−53) and C- (110−135) terminal regions of Pirt were generated by PCR amplification of Pirt
cDNA and cloned into pCMV-GST (Tsai and Reed, 1997
) in frame with glutathione S-transferase (GST). The mutant forms of the Pirt C terminus were generated using a QuikChange Site-Directed Mutagenesis Kit (Stratagene).
In Situ Hybridization
Nonisotopic in situ hybridization on frozen sections from WT E14.5 embryos was performed as previously described using cRNA probes (Dong et al., 2001
Rabbit polyclonal antibody was raised against a synthetic peptide, EVLPKALEVDERSPESKDL, corresponding to the N terminus of mouse Pirt by Protein-tech Group, Inc.
DRG neurons were collected from all spinal levels from 3- to 4-week-old mice and then were dissociated and plated on coated glass coverslips. Cells were plated for 24 hr before use. TRPV1 and TRPA1 stably expressing HEK cells were obtained from Drs. M. Caterina (Johns Hopkins University) and N. Tigue (GlaxoSmithKline), respectively. For transient transfection of HEK293 cells, Lipofectamine (Invitrogen) was used. Details of Neuronal and HEK cell culture conditions and transfection are available (see Supplemental Data
HEK293 cells were transfected with a Pirt-myc fusion construct along with a TRPV1, TRPM8, or TRPA1 plasmid. After 24 hr, cell lysates were collected and supernatant was extracted. Myc antibody was added to the supernatant and incubated overnight. Gammabind Plus Sepharose beads (GE Healthcare Life Sciences) were added to the lysates, and the mixtures were incubated for an additional 3 hr and the proteins were eluted using 2× SDS-PAGE sample buffer (see Supplemental Data
GST Pull Down
The GST-N, GST-C fusion, and GST constructs were separately transfected with various ion channel genes into HEK293 cells. After 24 hr, cell lysates were incubated using glutathione-agarose beads. Specifically bound proteins were eluted from the beads using 2× protein sample buffer. Details are available (see Supplemental Data
Biotinylation of Cell-Surface Proteins
TRPV1 stably expressing HEK293 cells were transfected with Pirt-containing plasmid. After 24 hr, the HEK cells or cultured DRG neurons were then incubated with 1.0 mg/ml sulfo-NHS-SS-Biotin (Pierce) in PBS for 30 min at 4°C. After quenching, cells were lysed in 500 μl of RIPA buffer. Supernatant from cell lysate was added to BSA-Blocked ultralink-neutravidin beads (Pierce) and incubated for 2 hr. The proteins were eluted from the beads using 2× SDS-PAGE sample buffer and western blot was performed using the anti-TRPV1 antibody (from Dr. M. Caterina). Details are available (see Supplemental Data
PIP2 Bead Binding and PIP Strips
HEK293 cells were singly transfected with GST-N, GST-C, or GST. The cells were lysed and spun down at 10,000 × g for 10 min. The supernatants were added to the PIP beads or PIP strips (Echelon Biosciences). Details are available (see Supplemental Data
Liposomal Binding Assay
Liposomes were prepared by mixing PC, PS, and PIPs at the proportions of 49.5%, 49.5%, and 1.0%, respectively, drying the mixture under vacuum, and resuspending to a final concentration of 1 mg/ml of total phospholipid in a buffer composed of HEPES (50 mM), NaCl (100 mM), and EDTA (0.5 mM). After sonicating for 30 min, liposomes were collected by centrifugation at 16,000 × g for 10 min and resuspended in binding buffer. Fifty microliters of liposome suspension was mixed with 0.1 μg of purified protein and incubated at room temperature for 15 min. Liposomes were pelleted at 16,000 × g for 10 min and bound fraction was analyzed by western blot (see Supplemental Data
Behavioral tests were performed by individuals blind to genotype. The mice used in the tests were backcrossed to C57Bl/6 mice for at least five generations and were 2- to 3 month-old (20−30 g) males. All experiments were performed under protocol approved by the Animal Care and Use Committee of Johns Hopkins University School of Medicine. Details for all the behavior assays are available in the Supplemental Data
Whole-cell voltage-clamp recordings were performed. For neurons, only small neurons were selected for recording. For HEK293 cells, transfected cells were identified by GFP fluorescence. Only one neuron or one HEK cell was tested per coverslip.
Capsaicin, mustard oil, and GABA were used as agonists to TRPV1, TRPA1, and GABA receptors, respectively. When examining heat-evoked current responses, a preheated solution was used to increase bath temperature; the heating was an in-line solution heater and used the TC-324B temperature controller (Warner Instruments). Statistical comparisons were made using un-paired Student's t test and differences were considered significant at p < 0.05. Details are available in the Supplemental Data