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Neuron. Author manuscript; available in PMC Jun 6, 2008.
Published in final edited form as:
Neuron. May 8, 2008; 58(3): 362–373.
doi:  10.1016/j.neuron.2008.04.012
PMCID: PMC2422846
NIHMSID: NIHMS50693

A Yeast Genetic Screen Reveals a Critical Role for the Pore Helix Domain in TRP Channel Gating

SUMMARY

TRP cation channels function as cellular sensors in uni- and multicellular eukaryotes. Despite intensive study, the mechanisms of TRP channel activation by chemical or physical stimuli remain poorly understood. To identify amino acid residues crucial for TRP channel gating, we developed an unbiased, high-throughput genetic screen in yeast that uncovered rare, constitutively active mutants of the capsaicin receptor, TRPV1. We show that mutations within the pore helix domain dramatically increase basal channel activity and responsiveness to chemical and thermal stimuli. Mutation of corresponding residues within two related TRPV channels leads to comparable effects on their activation properties. Our data suggest that conformational changes in the outer pore region are critical for determining the balance between open and closed states, providing evidence for a general role for this domain in TRP channel activation.

INTRODUCTION

Members of the transient receptor potential (TRP) family of nonselective cation channels participate in a wide variety of physiological processes in organisms ranging from fungi to humans (Clapham, 2003; Dhaka et al., 2006). TRP channels respond to such diverse agonists as hypertonicity in yeast, light-evoked phospholipase C (PLC) activation in the Drosophila photoreceptor, and noxious chemical and thermal stimuli in the mammalian somatosensory system. Accordingly, spontaneous loss- and gain-of-function mutations in TRP channels underlie disease states resulting from decreased or increased cation influx, respectively (Nilius, 2007). TRP channels belong to the six-transmembrane-domain (6TM, S1-S6) cation channel superfamily that includes depolarization-activated K+, Na+, and Ca2+ channels, as well as cyclic-nucleotide-gated (CNG) and hyperpolarization-activated (HCN) channels. TRP channels fall into several subfamilies, including vanilloid (V), canonical (C), and melastatin (M) groups, with most members suspected to form tetramers in vivo. Based on hydropathy predictions, each subunit contains intracellular N- and C-termini and a membrane-reentrant pore loop between S5 and S6. As a class, TRP channels bear resemblance at the level of transmembrane topology but show remarkably little primary sequence homology within and across subfamilies (Clapham, 2003).

TRP channels respond to an array of stimuli, including physical and chemical agonists (Clapham, 2003; Dhaka et al., 2006). For example, TRPV1, the founding member of the vanilloid subfamily, is the receptor for capsaicin (the pungent ingredient in “hot” chili peppers), but also responds to extracellular protons, bioactive lipids, and noxious heat (>43ºC) (Caterina et al., 1997; Jordt et al., 2003; Tominaga et al., 1998). Like many other members of the TRP channel family, TRPV1 can also be modulated by PLC-coupled signal transduction pathways (Chuang et al., 2001; Lukacs et al., 2007; Prescott and Julius, 2003). However, the molecular mechanisms underlying TRP channel activation, ranging from stimulus detection to the regulation of channel opening (gating), are poorly understood. This stands in contrast to other 6TM channels, most notably voltage-activated K+ channels, which have been probed in great detail using biophysical and crystallographic methods (Tombola et al., 2006; Yellen, 1998).

For voltage-gated channels, an important clue to their common activation mechanism came from visual inspection of linear protein sequences, which revealed a discrete domain (the positively charged S4 helix) as a likely universal candidate for translating changes in plasma membrane voltage into channel opening (Tombola et al., 2006; Yellen, 1998). In contrast, TRP channels are activated by a diversity of chemical and physical stimuli for which corresponding functional motifs are not readily apparent in the channel polypeptide (Clapham, 2003), making it challenging to gain insight into the gating process. Furthermore, TRP and voltage-gated channels are not only functionally divergent, but also show sparingly little sequence similarity. Thus, the use of previously developed structural and mechanistic models to ascertain TRP channel function is rather limited. This leaves unanswered the important question of whether any unifying mechanistic principles underlie TRP channel gating.

While previous reports have delineated molecular determinants for agonist binding and activation in several TRP family members, including TRPV1 (Jordt and Julius, 2002; Jordt et al., 2000; Jung et al., 2002), few if any conserved structural elements controlling TRP channel gating have been reported. The difficulty in pinpointing critical gating domains stems, in part, from the challenge of conducting mutational analyses that efficiently sample a large sequence space. Conventional ion channel structure-function studies generally entail the construction and testing of channel mutants one at a time; such studies are low-throughput, precluding an unbiased examination in which multiple substitutions at each sequence position are evaluated for functional alterations. Some studies have exploited amino acid polymorphisms between functionally divergent TRP channel family members to narrow down blocks of sequence that contain determinants of agonist activation (via analysis of chimeric channels) (Chuang et al., 2004; Jordt and Julius, 2002), but this methodology is poorly suited to uncovering core structural elements which are expected to show high levels of conservation among TRP channels. Such limitations can be partially overcome by using robotic methods to sample large numbers of individual mutants via high-throughput functional assays (Bandell et al., 2006; Decaillot et al., 2003; Zhou et al., 2007). Alternatively, genetic strategies based on simple life-or-death readouts (Loukin et al., 1997; Minor et al., 1999; Ou et al., 1998) could permit selection of functionally interesting channel mutants, but such a methodology has not been applied to mammalian TRP channels.

Here we describe the identification and characterization of a critical component of the TRP channel gating machinery via unbiased genetic screening of a randomly generated population of TRPV1 mutants. We conducted this high-throughput screen on mammalian TRPV1 heterologously expressed in the budding yeast Saccharomyces cerevisiae, whose superior genetics allows for the efficient isolation of constitutively active channel alleles. Our study reveals that gain-of-function substitutions in the pore helix of TRPV1 dramatically affect channel activation, establishing an important role for this domain in TRPV1 functionality. We extend these observations to two other members of the TRPV subfamily, suggesting that the pore helix is a key regulatory domain that plays a conserved role in the gating of at least a subset of excitatory TRP channels.

RESULTS

Functional Expression of the Capsaicin Receptor in Yeast

Persistent activation of native or recombinant vanilloid receptors leads to a dramatic decrease in mammalian cell viability due to overload with cations (Caterina et al., 1997; Jancso et al., 1977; Wood et al., 1988). We reasoned that if TRPV1 forms functional channels upon heterologous expression in yeast, then exposure to capsaicin should be fatal. Indeed, yeast transformed with TRPV1 grew robustly on standard solid medium, but failed to grow on medium containing capsaicin (Fig. 1A). Capsaicin-evoked toxicity was concentration dependent with half-maximal death occurring in the high nanomolar range (data not shown), consistent with established dose-response profiles in mammalian cells (Gunthorpe et al., 2000; Jerman et al., 2000; Oh et al., 1996). In contrast, capsaicin did not change the viability of a strain harboring a Kir2.1 inward rectifier K+ channel (Minor et al., 1999) (Fig. 1A), suggesting that capsaicin-evoked toxicity is mediated by TRPV1 and is not simply an artifact of expressing a foreign cation channel. Along these lines, we found that lethality was fully suppressed by a TRPV1 pore blocker, ruthenium red (10 μM) (Fig. 1A), or significantly attenuated by an inverse agonist, capsazepine (50 μM) (data not shown). We have previously described a hypersensitive TRPV1 mutant in which an inhibitory C-terminal putative PIP2 binding module has been deleted, leading to activation at sub-threshold agonist concentrations or ambient temperatures (Prescott and Julius, 2003). Consistent with such functional properties, we found that expression of this potentiated mutant (TRPV1 Δ777-820) was lethal to yeast even in the absence of capsaicin, but growth was restored to wild type levels in the presence of ruthenium red (Fig. 1B). Furthermore, we found that the lethality of capsaicin was augmented when the yeast medium was supplemented with higher levels of sodium (data not shown), suggesting that, as in mammalian cells, toxicity stems from a rise in intracellular cation concentrations to intolerable levels.

Figure 1
Mammalian TRPV1 forms functional channels in yeast

Our growth assay demonstrates that sustained TRPV1 activation produces similar long-term effects on survival of yeast and metazoan cells. Furthermore, activation of TRPV1 in yeast by thermal or chemical stimuli occurs on a physiologically relevant time scale, shown by visualizing TRPV1-mediated cobalt accumulation as a direct, short-term assay of channel activity. Stimulus-dependent uptake of cobalt chloride into TRPV1-expressing cells is observed following addition of ammonium sulfide, which generates a readily visible black cobalt sulfide precipitate (Winter, 1987; Wood et al., 1988). In TRPV1-expressing yeast, pellets from non-stimulated cells appeared off-white, but became deep black within five minutes of capsaicin treatment (Fig. 1C), yielding a dose-response relationship consistent with previous functional assays (EC50 ~ 70 nM) (Fig. 1D). As in the cell death assay, Kir2.1-transformed cultures did not respond to capsaicin, and capsaicin stimulation of TRPV1 in absence of cobalt produced no color change. Similarly, TRPV1-expressing yeast responded robustly to a rise in bath temperature in a manner that closely parallels heat-evoked channel activation in electrophysiological studies (Fig. 1C, E) (Caterina et al., 1997; Tominaga et al., 1998). Thus, our cobalt uptake assay illustrates that TRPV1 forms functional channels in yeast that are reminiscent of native or cloned channels expressed in vertebrate cells.

Unbiased Forward Genetic Screen Reveals Gain-of-Function Mutations

Constitutively active TRPV1 mutants might harbor deficits in some aspect of channel activation, and a comprehensive list of such mutations could provide valuable information about the location of the channel gate. We used the activity-dependent death phenotype described above to identify such TRPV1 alleles which, unlike the wild type channel, conferred significant toxicity even under basal conditions (i.e., in the absence of capsaicin at 30°C). To execute this screen, we introduced a library of randomly mutagenized TRPV1 cDNAs into yeast and plated transformants onto medium containing ruthenium red, thereby blocking TRPV1 to allow for growth of all cells independent of channel activity. Resulting colonies were replica plated onto medium lacking ruthenium red to unmask the lethality of any constitutively active channels (Fig. 2A). Colonies showing reduced survival in the absence of ruthenium red were picked from the original plate and retested to confirm their phenotype.

Figure 2
Yeast genetic screen identifies gain-of-function TRPV1 alleles

From this screen, we recovered TRPV1 alleles showing a range of toxic effects (Fig. 2B). Out of approximately 46,000 clones examined, 46 reproducibly exhibited growth impairment in the absence of ruthenium red, yielding an overall hit rate of 0.1% (Table S1). This is in reasonable agreement with the hit rate obtained from a luminometry-based screen for gain-of-function alleles in the yeast TRP channel Yvc1p (Su et al., 2007; Zhou et al., 2007), and is consistent with the general principle that gain-of-function mutations in channels are rare. Our set of TRPV1 gain-of-function alleles defined a total of 30 unique mutations at 25 amino acid positions (Table S2). Interestingly, recovered mutations were clustered within the presumptive pore and cytoplasmic termini (Fig. 2C). Only two gain-of-function mutations were recovered within the first four transmembrane domains, even though sequencing of randomly selected library clones showed equal mutagenic frequency throughout the coding region. Given the architectural similarity between TRP and voltage-gated channels and the critical role played by the first four transmembrane helices in voltage sensitivity, we were surprised to observe a relative paucity of gain-of-function alleles within this module.

Electrophysiological analysis reveals a range of functional alterations

We used electrophysiological methods to directly characterize the functional properties of mutant channels when expressed in Xenopus oocytes. For all channels, basal and proton-evoked currents were assessed at pH 7.4 and 6.4, respectively, and normalized to a saturating concentration (10 μM) of capsaicin. As expected, wild type TRPV1 responded strongly to capsaicin, but displayed negligible current under basal conditions or in response to pH 6.4 (Fig. 3A), which is just at the threshold concentration required for proton-evoked activation at room temperature (Chuang et al., 2001; Jordt and Julius, 2002; Tominaga et al., 1998). In contrast, eight of the recovered mutants displayed a significant response to pH 6.4, although no basal current was detected at pH 7.4 (Fig. 3B, E). We labeled such mutants as “potentiated.” Furthermore, a second group of mutants displayed a more extreme phenotype, manifest as large basal currents at pH 7.4 (≥ 15% of the maximum capsaicin-evoked response) (Fig. 3C, D, E). These basal currents showed characteristic outward rectification and block by ruthenium red, indicating that they are carried by TRPV1. We refer to these mutants as “constitutively active.”

Figure 3
Mutants recovered from the yeast screen display altered electrophysiological properties

Among the constitutively active channels, residues K155 and K160 map to a single N-terminal ankyrin repeat domain, while M581T and F640L are in the S5-pore-S6 region (Fig. 2C). Interestingly, the two N-terminal lysine residues have been previously shown to bind cytoplasmic ATP and calmodulin (Lishko et al., 2007). Lysine-to-alanine substitutions at these positions abolish interaction with ATP or calmodulin and reduce tachyphylaxis (the process by which repeated channel stimulation evokes progressively weaker responses), thereby decreasing efficiency of channel closure upon repeated stimulation. These mutants also display hypersensitivity to capsaicin (Lishko et al., 2007). We asked whether the glutamate substitutions identified by our screen are mechanistically equivalent to the previously described alanine substitutions. Indeed, both sets of substitutions led to comparable toxicity in yeast (Fig. S1A) and high basal currents in oocytes (Fig. S1B), presumably reflecting constitutive activity and/or a failure to desensitize. In any case, our independent identification of these mutations validates the ability of the yeast screen to pinpoint functionally significant TRPV1 residues.

In summary, three functionally distinct classes of TRPV1 alleles arose from our screen, ranging from mutations causing little perturbation of basal or stimulus-evoked activity to those causing dramatic constitutive activation. Interestingly, not all mutants arising from the screen exhibit marked electrophysiological phenotypes in oocytes. This disparity could reflect physiological differences between yeast and metazoan systems, such as transmembrane voltage (highly negative in yeast), growth temperature and extracellular pH, or differential expression of regulatory factors. Nonetheless, the screen has clearly enriched for mutants with heightened agonist sensitivity, consistent with our goal of identifying substitutions rendering TRPV1 constitutively active or hypersensitive.

F640L renders channels hypersensitive to thermal and chemical stimuli

Among the mutants displaying high constitutive activity in oocytes, we focused on F640L for three main reasons. First, F640L displayed the strongest basal channel activation of all mutants examined (Fig. 3C, D), suggestive of a dramatic alteration in TRPV1 function. Second, F640 lies within the presumptive TRPV1 outer pore, a region that remains relatively unexplored with regard to TRP channel gating. Finally, the F640L allele displayed a unique phenotype in regard to basal versus proton-evoked currents, as described in more detail later.

We examined the biophysical properties of the F640L mutant by patch-clamp recording of transfected HEK293 cells. Consistent with its yeast phenotype, the F640L mutant conferred substantial toxicity when expressed in HEK293 cells, characterized by necrotic morphology similar to that observed in cells expressing wild type TRPV1 after prolonged exposure to capsaicin (Caterina et al., 1997) (Fig. 4A, B). Inclusion of ruthenium red (3 μM) in the culture medium significantly attenuated the death of F640L-expressing cells, permitting electrophysiological analysis.

Figure 4
F640L mutant TRPV1 channels are toxic and display high basal activity in mammalian cells

We examined the sensitivity of F640L mutant channels to chemical and thermal stimuli. Inside-out membrane patches from HEK293 cells expressing wild type TRPV1 showed small but measurable basal activity at room temperature that exhibited strong outward rectification (i.e. negligible inward current at −60 mV), consistent with previous reports (Chuang et al., 2001; Matta and Ahern, 2007). Subsequent challenge of these cells with a saturating dose of capsaicin (10 μM) elicited a large (approximately 200-fold) induction of the basal current (Fig. 4C). In contrast, inside-out patches from the F640L mutant routinely displayed large basal currents with a substantial inward component (Fig. 4D). Despite this difference in basal activity, addition of capsaicin at saturating concentrations produced currents of similar magnitude to those evoked in patches containing wild type channels. We found no significant difference in either the single channel conductance or the relative permeabilities for Na+, K+, and Ca2+ ions when comparing wild type and F640L mutant channels (Figure S2), showing that the F640L mutation affects gating rather than permeation properties.

Consistent with a hypersensitive gating mechanism, we found that the F640L mutant displayed a 35-fold leftward shift in the capsaicin dose-response curve compared to the wild type receptor (EC50 = 1.91 ± 1.26 and 65.62 ± 4.03 nM, respectively) (Fig. 5A). Interestingly, mutant channels also exhibited a lower Hill coefficient (0.33 ± 0.08 versus 2.06 ± 0.20 for wild type TRPV1), indicative of decreased agonist cooperativity. Importantly, the basal F640L-mediated current was suppressed by the inverse agonist capsazepine (Fig. 5D), demonstrating that the high constitutive activity is not due to an inability of the channel to close. This mutant also exhibited robust currents well below room temperature, corresponding to a dramatic shift in thermal activation threshold (Fig. 5B) which resembles a proton-potentiated response in cells expressing wild type TRPV1 (Jordt et al., 2000; Tominaga et al., 1998). Because the basal responsiveness of the F640L mutant was reduced with decreased ambient temperature, the mutant phenotype is unlikely to result from a defect in temperature detection per se, but rather from an independent mechanism that more generally affects gating. Our data suggest that the gating machinery remains intact in the F640L mutant, but the equilibrium has shifted to favor the open state.

Figure 5
F640L TRPV1 mutant channels show enhanced chemical and thermal sensitivity, but decreased proton potentiation

F640L mimics and occludes proton-mediated channel potentiation

In contrast to all other mutants in the high constitutive activity group, only the F640L basal currents were not further potentiated under low pH conditions (Fig. 3E). Nonetheless, addition of capsaicin to F640L-expressing cells led to a marked increase in current, demonstrating that F640L channels are not maximally open in the basal state (Fig. 3C, ,4D).4D). As such, the failure to observe proton-evoked potentiation likely reflects a perturbation in the gating mechanism through which protons exert their physiological regulatory effect on TRPV1.

In mammalian cell patch-clamp experiments, we found that, although the wild type channel was potentiated by exposure to pH 6.2, the F640L current was completely unaffected under these conditions (Fig. 5C–E), demonstrating a defect in proton-evoked potentiation. We also observed that a weakly alkaline solution (pH 8.2) failed to reduce the F640L-mediated basal current, even though the same conditions suppressed currents evoked by a moderate dose of capsaicin (70 nM) in cells expressing the wild type channel (data not shown). However, higher doses of protons could activate the F640L mutant in a manner similar to the wild type channel (pH50 = 5.60 ± 0.03 versus 5.72 ± 0.19 for wild type and F640L mutant channels, respectively), illustrating that while the mutant has lost the ability to be potentiated within a certain pH range, its proton activation has not been completely ablated.

Taken together, our data show that the F640L mutation enhances sensitivity to heat and capsaicin by shifting the stimulus-response relationships of the channel leftward, while also decreasing apparent cooperativity of gating. Indeed, this phenotype closely resembles that of wild type TRPV1 channels operating under acidic conditions. In contrast, the sensitizing effects of extracellular protons are lost in the F640L channel, as one might expect if this mutation phenocopies the proton potentiated state.

Additional substitutions in the outer pore alter TRPV1 gating

The F640 residue is located near the C-terminal end of a 15 amino acid stretch immediately preceding the presumptive selectivity filter, a region known in K+ channels as the pore helix (Doyle et al., 1998). While TRP and K+ channels share limited homology, computer algorithms (Jones, 1999) predict a high degree of helicity in this region of TRPV1, suggesting that a pore helix may indeed be present (Fig. 6A). While it is generally accepted that the outer pore region contains structural determinants for ion permeation and block (Yellen, 1998), the role of pore helices in the regulation of channel gating has been less fully explored. Our yeast screen and functional characterization of F640L suggest that the pore helix may be a particularly critical for TRPV1 gating.

Figure 6
Additional substitutions in the TRPV1 pore helix affect channel activation

We randomized the F640 codon to fully explore the structural requirements at this position (Fig. 6B). Transformants from a F640 mini-library were evaluated for wild type, gain of function, or loss-of-function behavior using the yeast growth assay. Most substitutions at this position, particularly those of a hydrophilic nature, weakened or abolished channel activity. In contrast, several hydrophobic amino acids supported wild type functionality, except for leucine and isoleucine, which produced constitutively active channels. Thus, our codon randomization illustrates that most hydrophobic substitutions at F640 produce functional channels whereas two small hydrophobic residues support constitutive channel activity, suggesting that F640 is buried in a non-polar environment.

Although we analyzed a large number of clones in our initial yeast screen, it is possible that some function-altering substitutions went undetected. We therefore screened a more restricted yeast library in which only the TRPV1 pore helix and its immediate environment (residues 626–660) were randomly mutagenized to uncover additional substitutions in this region that might lead to constitutive activation (Table S1). We uncovered nine additional substitutions conferring a toxic phenotype in yeast. Of these, two (N628D and V658A) showed a potentiation effect under moderately acidic conditions, while two more (T641S and T650S) showed high basal activity (Fig. 6C, D). Interestingly, T641S (and, to a lesser extent, T650S) mutants displayed large constitutive channel activation with relative insensitivity to pH 6.4. The T641S mutant also caused widespread death in HEK293 cells (data not shown). Thus, at least two mutants recovered in the outer pore region are reminiscent of the original F640L mutation. Because substitutions in two adjacent residues (F640 and T641) produce similar functional alterations, we conclude that the short stretch of amino acids at the interface between the pore helix and the permeation pathway forms a particularly crucial part of the gating apparatus.

The functional role of the pore helix is conserved in related TRP family members

Mammalian TRPV channels are highly divergent at the amino acid level. Rat TRPV1, TRPV2, TRPV3, and TRPV4 exhibit only 21% identity overall, while TRPV5 and 6 are even more distantly related. Interestingly, the 13 residue-long stretch connecting the pore helix and selectivity filter displays 69.2% identity in TRPV1 – 4 (Fig. 6A), suggesting that some TRP channels may share common architectural features within this region. We therefore asked whether any pore helix mutations identified in our TRPV1 screen would produce a gain-of-function effect in other TRPV family members.

TRPV3 is activated by warm temperatures, with a threshold in the 33°C – 38°C range (Peier et al., 2002; Smith et al., 2002; Xu et al., 2002). Sequence alignments reveal a threonine at position 636 in TRPV3 corresponding to T641 in TRPV1 (Fig. 6A). We asked whether TRPV3 T636S channels displayed constitutive activity similar to TRPV1 T641S. Strikingly, transfection of TRPV3 T636S into HEK293 cells produced massive cell death that was significantly attenuated by ruthenium red, while no toxicity was observed from the wild type channel (Fig. 7D). In inside-out patches excised from oocytes expressing wild type TRPV3, we observed small outwardly rectifying basal currents that were robustly stimulated by the broad spectrum TRP channel agonist 2-aminoethoxydiphenyl borate (2-APB) (Chung et al., 2004a; Hu et al., 2004) (Fig. 7A). In contrast, TRPV3 T636S exhibited large basal currents characterized by high channel noise and outward rectification (Fig. 7B), reminiscent of wild type TRPV3 activity at warm temperatures. As with the TRPV1 pore helix mutant, TRPV3 T636S channels showed substantial basal currents at negative holding potentials. By normalizing the basal currents to a maximal 2-APB-evoked response (300 μM), we estimate that T636S displays approximately 300-fold increase in spontaneous activity compared to the wild type channel (Fig. 7C). TRPV3 T636S basal currents were suppressed by cold (10°C), and the inward current was strongly attenuated by ruthenium red (Fig. 7A–B, and data not shown), a pharmacology consistent with TRPV3-evoked currents in native or heterologous systems (Chung et al., 2004b; Peier et al., 2002; Smith et al., 2002; Xu et al., 2002). We also investigated the effects of an F603L substitution in TRPV2, a position corresponding to F640L in TRPV1. Unlike other TRPV channels, wild type TRPV2 exhibits no measurable basal activity when heterologously expressed, and we did not detect basal currents from either wild type or F603L TRPV2 channels (data not shown). Nevertheless, we observed that the F603L mutant displays enhanced 2-APB sensitivity (Fig. 7E, F). Taken together, our data show that mutations in pore helices shift multiple TRPV channels into an open conformation, showing that this region plays a conserved role in gating of different TRP family members by diverse chemical and thermal stimuli.

Figure 7
The gating function of the pore helix is conserved across TRPV channels

DISCUSSION

Conservation of TRPV1 functional properties in a non-mammalian genetic system

Spontaneous gain-of-function mutations have provided valuable insights into ion channel permeation and gating (Kohda et al., 2000; Navarro et al., 1996). While naturally occurring mutations have been identified in mammalian TRP channels (Asakawa et al., 2006; Kim et al., 2007; Xu et al., 2007), these sporadic genetic events are too rare to support a systematic analysis of TRP channel functionality. Here we report a facile and unbiased forward genetic screen for isolating gain-of-function mutations in a mammalian TRP channel based on a simple yeast growth phenotype. This high-throughput methodology enabled us to survey the TRPV1 coding region for substitutions that cause constitutive activation. Such an approach has been applied to microbial cation channels (Loukin et al., 1997; Ou et al., 1998) and vertebrate inward rectifier K+ channels (Sadja et al., 2001; Yi et al., 2001), and we now extend this strategy to include non-selective mammalian cation channels. Previous studies have exploited polymorphisms between TRP channel homologues or orthologues to delineate molecular determinants underlying ligand activation (Chuang et al., 2004; Jordt and Julius, 2002; Ryu et al., 2007), but this methodology is limited for numerous reasons. TRP channel subtypes generally exhibit low sequence homology and thus channel chimeras are often non-functional. In addition, such an approach cannot pinpoint residues that are conserved across channels. Indeed, many mutations uncovered by our screen are highly conserved in TRPV1 orthologues or other TRPV family members, attesting to the increased power of this approach for detecting core structural elements. A select percentage (0.10%) of mutant channels emerged from the cell death assay, of which ~40% showed bona fide functional perturbations when examined in vertebrate expression systems, attesting to both the stringency and efficiency of the screen. Perhaps most importantly, this screen revealed a number of interesting channel mutants that have not been described in previous structure-function studies.

Remarkably, rat TRPV1 expressed in yeast recapitulates functional properties observed in neurons or other mammalian cell types, including sensitivity to noxious heat and capsaicin. This suggests that both chemical and thermal sensitivity are intrinsic channel properties, not requiring membrane or cytoplasmic factors specific to metazoans. Moreover, yeast are hyperpolarized compared to metazoan cells, with membrane potentials estimated at −100 to −250 mV (Serrano and Rodriguez-Navarro, 2001). The observation that TRPV1 can be robustly activated in yeast therefore favors a modest role for membrane voltage as a regulator of channel gating (Latorre et al., 2007; Matta and Ahern, 2007).

The pore helix as a transducer of TRPV1 gating

A hallmark of many TRP channels is their ability to integrate multiple physiological inputs (Clapham, 2003; Dhaka et al., 2006; Julius, 2005). This is exemplified by TRPV1, whose role as a polymodal detector of chemical and physical stimuli underlies its capacity to regulate sensory neuron excitability under normal and pathophysiological conditions (Tominaga et al., 1998). Different TRPV1 stimuli (e.g. capsaicin, heat, acidic pH) produce synergistic effects on channel activation, while antagonists (e.g. capsazepine, cold, alkaline pH) diminish responses to multiple activating stimuli, providing evidence for crosstalk and convergence in channel gating mechanisms (Tominaga et al., 1998). This is consistent with our observation that the F640L mutant shows a shallowing of dose-response profiles to capsaicin and temperature, accompanied by a leftward shift in activation thresholds. This decreased cooperativity and increased sensitivity suggest that the pore helix region undergoes a structural transition common to all activating modalities, for which the energetic barrier is lowered by mutations such as F640L.

In the case of extracellular protons, it is possible to separate their ability to potentiate other stimuli under moderately acidic (pH 6.4) conditions versus their ability to activate TRPV1 de novo at room temperature (pH < 6.0). Mutations at two extracellular glutamate residues, E600 or E648, selectively alter the modulatory versus activating effects of protons, respectively (Jordt et al., 2000). In this regard, it is interesting that F640L displays agonist hypersensitivity and loss of proton-mediated potentiation, but shows normal de novo gating by protons, similar to positively charged substitutions at E600 (Jordt et al., 2000). Thus, perturbations in the pore helix are sufficient to favor an open conformation, perhaps mimicking and occluding the modulatory effect mediated by interaction of protons with extracellular sites such as E600.

While our data establish a role for the pore helix in proton-mediated potentiation, recent work suggests that this domain may also be involved in de novo proton gating. Mutation of residue T633 in TRPV1, also located within the pore helix, abolishes proton-evoked activation, although protons can still potentiate currents evoked by capsaicin (Ryu et al., 2007). Thus, other pore helix substitutions show a distinct but related phenotype to that observed in our study, namely, alteration of de novo proton-evoked activation rather than proton-mediated potentiation. These studies support the general conclusion that conformational changes in the pore helix are critical for both the activating and modulatory effects of protons on TRPV1, and underscore the idea that, while proton-mediated potentiation and de novo activation are determined by distinct sets of residues, their effects on channel gating both involve the pore helix as a key regulatory domain.

Based on secondary structure predictions and their relationship to known K+ channel structures, E600 and E648 are predicted to lie near the extracellular face of the outer pore, close to the top of the pore helix (Fig. 8), and it is conceivable that protonation of these residues could induce conformational rearrangements propagating through the pore helix that ultimately lead to channel opening. Perhaps the C-terminus of the pore helix forms a movable barrier to ion flux, either by protruding directly into the permeation pathway or by constricting the selectivity filter into a nonconductive conformation similar to that observed in one crystal structure of the prokaryotic K+ channel KcsA (Zhou et al., 2001). Alternatively, residues within the pore helix might not form a physical occlusion per se, but rather stabilize an open conformation of the channel, comparable to a model proposed for the gating of inward rectifier K+ channels (Alagem et al., 2003).

Figure 8
A structural model for the pore region of TRP channels

The pore helix as a conserved element in TRP channel gating

Our present analysis of the pore helix suggests that its role in gating is conserved in at least a subset of TRP channels. We found that mutation of pore helix residues leads to constitutive activation of TRPV3, as well as potentiation of TRPV2. Therefore, our study suggests that the pore helix plays a general role in TRP channel gating that has not been previously appreciated. TRPV1 may represent a case in which a universal gating element has been usurped to serve as an allosteric regulatory site for protons, allowing for alteration of TRPV1 sensitivity in the setting of inflammation and tissue acidosis. Thus far, relatively few structural elements have been shown to play conserved functional roles in TRP channels, and it may be worthwhile to investigate pore helices as gating elements in other mammalian TRP family members.

While numerous studies have localized the molecular determinants of ion permeation and block to the outer pore region of TRP (Voets et al., 2004; Voets et al., 2002) and other cation channels (Ahern et al., 2006; Chatelain et al., 2005; Doyle et al., 1998), the role of the pore helix in TRP channel gating and agonist sensitivity is unexpected. Indeed, structural analyses suggest that voltage activation of K+ channels involves alteration of interactions between the S4–S5 linker and the cytoplasmic end of S6, relieving strain on the helical bundle crossing that forms the intracellular gate (Long et al., 2005a, b; Tombola et al., 2006). In this model, the outer pore region is an ion conduit that remains relatively stationary during the overall gating transition. On the other hand, the outer pore is known to play a critical role in the context of C-type inactivation, the process by which repetitive voltage stimulation desensitizes voltage-gated K+ channels via a mechanism involving collapse of the selectivity filter. Mutations in the outer pore region, including the pore helix, can dramatically influence the extent of K+ channel C-type inactivation, suggesting that these regions play an important role in modulating the biophysical properties of open K+ channels (Cordero-Morales et al., 2006; Hoshi et al., 1991; Liu et al., 1996; Lopez-Barneo et al., 1993). Thus, the outer pore can influence the level of K+ channel activity under certain conditions.

Aside from K+ channels, additional studies have suggested a role for the pore helix in gating of other 6TM channels. Cysteine accessibility experiments in CNG channels (Liu and Siegelbaum, 2000), as well as in TRPV5 and TRPV6 (Voets et al., 2004; Yeh et al., 2005), show that residues in the pore helix are differentially reactive to modifying agents in the closed and open states, illustrating that in these cases (and unlike K+ channels), the pore helix may rotate during gating. Together with our findings, these observations indicate that, despite topological resemblance to voltage-gated K+ channels, gating of other members of the 6TM superfamily may involve a conformational change in the outer pore region. This is not to say that the structural determinants of activation identified in voltage-gated channels are irrelevant to TRP channels. Indeed, recent work has identified spontaneous dominant mutations in TRPV3 linked to hair loss and atopic dermatitis that dramatically enhance basal activity when engineered into the cloned channel (Asakawa et al., 2006; Xiao et al., 2007). Interestingly, both mutations map to a single residue in the putative S4–S5 region of TRPV3, suggesting that conformational changes in this linker might also play a role as molecular determinants of channel activation, perhaps via interaction with a cytoplasmic activation gate at the intracellular segment of S6. These and other studies highlight the idea that TRP channel gating likely involves multiple regions including the pore helix and the S4–S5 linker. Future work will address the physical and functional interplay between these regions in shaping the physiological properties of TRP channels.

EXPERIMENTAL PROCEDURES

Molecular biology

Rat TRPV1 was cloned into p425GPD. For mutagenic library construction, TRPV1 was amplified using Taq polymerase (New England Biolabs) under standard conditions for 25 cycles, and the mutagenized N- or C-terminal cassettes separately reintroduced back into the wild type construct to generate two distinct libraries with the desired mutagenesis frequency. For the pore helix library, mutagenesis rate was boosted by supplementing the PCR with MgCl2 and MnCl2 (final concentrations of 7 and 0.5 mM, respectively), increasing cycle number to 40, and decreasing concentration of dATP and dGTP relative to dCTP and dTTP. Site-directed mutagenesis was performed by overlap extension PCR or QuikChange (Stratagene), or by yeast in vivo recombination. Mutant channels were cloned into the mammalian/oocyte expression vector pMO (gift of Lily Jan) for functional analysis. All constructs were verified by DNA sequencing.

Yeast transformation and replica plating

Strain BY4741 (Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was transformed with lithium acetate/polyethylene glycol and selected on medium lacking leucine. Media was supplemented with 20 mM MES and buffered to pH 6.2, under which conditions little or no inward current is observed in mammalian cells expressing TRPV1 (Figure 5C). Capsaicin (Tocris) and/or ruthenium red (Sigma) were added to molten agar prior to pouring. After two days growth on selective medium with 3 μM ruthenium red (mother plate), transformants were replica plated onto two plates, one lacking and another containing 10 μM ruthenium red (daughter plates), the latter designed to eliminate false positives arising from inefficient colony transfer from the mother plate. After two more days, the daughter plates were aligned to identify clones exhibiting basal toxicity. Clones from this first round of screening were retested by serial dilution. Plasmids were subsequently rescued and retransformed into fresh BY4741 to confirm the phenotype.

Cobalt uptake assay

Yeast cultures were grown to mid-log phase in selective medium and washed once in cobalt uptake assay buffer (in mM: 58 NaCl, 5 KCl, 2 MgCl2-6H20, 0.75 CaCl2, 12 glucose, 137 sucrose, 10 HEPES, pH 7.4). Pellets were resuspended in assay buffer with 5 mM CoCl2 plus varying concentrations of capsaicin and incubated for 5 min at 30°C. For temperature experiments, cells were subjected to varying temperatures for five minutes using a gradient PCR thermocycler (MJ Research). Cells were washed twice in assay buffer and stained with 1% (NH4)2S.

Mammalian cell culture and electrophysiology

HEK293T cells were cultured and transfected as previously described (Chuang et al., 2004.) For some experiments, we generated stable tetracycline-inducible Flp-in T-rex HEK293 cell lines (Invitrogen) expressing wild type TRPV1 or the F640L mutant. For whole-cell or excised patch recordings, bath solution contained (in mM): 140 NaCl, 10 HEPES, 1 MgCl2-6H2O, 1 EGTA, pH 7.4. Pipette solution was identical, except that CsCl was substituted for NaCl. For proton experiments, we substituted MES for HEPES in the bath and adjusted pH accordingly. For single channel analysis, pipette solution contained 1 μM capsaicin. Relative permeabilities were estimated from reversal potential shifts between solutions containing (in mM): 10 HEPES, 10 glucose, and either 140 NaCl, 140 KCl, or 125 NMDG-Cl + 10 CaCl2. Liquid junction potentials did not exceed 3 mV. Pipettes were fabricated from borosilicate glass (WPI) with resistances after fire-polishing of 1–2 MΩ for whole-cell experiments, 0.8–1.2 MΩ for inside-out macropatch experiments, and 6–10 MΩ for cell-attached single channel experiments. Currents were recorded with an Axopatch 200B amplifier (Molecular Devices) using a 180 ms voltage ramp from −120 mV to +80 mV delivered once per second. Currents were recorded at 5 kHz, filtered at 2 kHz, and analyzed with pClamp 10 (Molecular Devices). Temperature ramps were generated with a custom-made Peltier device (Reid-Dan Electronics). Capsaicin, capsazepine, and 2-APB were dissolved in DMSO, and ruthenium red was dissolved in water. Drugs were diluted into recording solution immediately prior to experiments.

Xenopus oocyte culture and electrophysiology

Surgically extracted oocytes from Xenopus laevis (Nasco) were cultured and analyzed 2–14 days post-injection by TEVC as previously described (Chuang et al., 2004). For proton experiments, MES was substituted for HEPES and pH adjusted accordingly. For patch clamp analysis, both the bath and pipette solutions contained (in mM): 140 CsCl, 10 HEPES, 1 MgCl2-6H20, 1 EGTA, pH 7.4. Macropatches were excised from devitellinized oocytes using pipettes with resistances of 0.3–0.8 MΩ and currents were analyzed as described for mammalian cell patch clamp.

Mammalian cell death assays

After 16 hours, transfected HEK293 cells were stained with DAPI (500 ng/ml) to identify dead cell nuclei. DAPI-positive cells were averaged from four different fields per transfection. Each field contained approximately the same total number of cells.

Supplementary Material

01

Acknowledgments

We thank members of the Julius lab for advice and encouragement at all stages of this project. We are especially grateful to D. Minor for valuable suggestions on establishing the yeast assays and H-h. Chuang, A. Priel, and Y. Kirichok for guidance on electrophysiological experiments. We thank H. Madhani, J. Weissman, and members of their labs for strains, plasmids, and assistance with yeast methods. We also thank R. Nicoll, D. Bautista, A. Chesler, D. Minor, and H-h. Chuang for insightful discussions and criticism of the manuscript. This work was supported by an American Heart Association Predoctoral Fellowship (B.R.M.), a UCSF Neuroscience NIH Training Grant (C.J.B.), and grants from the National Institutes of Health (NINDS) (D.J.).

Footnotes

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