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Am J Physiol Cell Physiol 291: C1022-C1028, 2006. First published July 5, 2006; doi:10.1152/ajpcell.00606.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Conserved cysteine residues in the pore region are obligatory for human TRPM2 channel function

Zhu-Zhong Mei, Hong-Ju Mao, and Lin-Hua Jiang

Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom

Submitted 5 December 2005 ; accepted in final form 16 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
TRPM2 proteins belong to the melastatin-related transient receptor potential or TRPM subfamily and form Ca2+-permeable cationic channels activated by intracellular adenosine diphosphoribose (ADPR). The TRPM2 channel subunit, like all its close relatives, is structurally homologous to the well-characterized voltage-gated potassium channel subunits, each containing six transmembrane segments and a putative pore loop between the fifth and sixth segments. Nevertheless, the structural elements determining the TRPM2 channel functions are still not well understood. In this study, we investigated the functional role of two conserved cysteine residues (at positions 996 and 1008) in the putative pore region of the human TRPM2 by site-directed mutagenesis, combined with electrophysiological and biochemical approaches. Expression of wild-type hTRPM2 channels in human embryonic kidney (HEK-293) cells resulted in robust ADPR-evoked currents. Substitution of cysteine with alanine or serine generated mutant channels that failed to be activated by ADPR. Furthermore, experiments done by Western blot analysis, immunocytochemistry, biotin labeling, and coimmunoprecipitation techniques showed no obvious changes in protein expression, trafficking or membrane localization, and the ability to interact with neighboring subunits that is required for channel assembly. Coexpression of wild-type and mutant subunits significantly reduced the ADPR-evoked currents; for the combination of wild-type and C996S mutant subunits, the reduction was ~95%, indicating that incorporation of one or more nonfunctional C996S subunits leads to the loss of channel function. These results taken together suggest that the cysteine residues in the pore region are obligatory for TRPM2 channel function.

ADP-ribose; site-directed mutagenesis; Western blot; patch-clamp recording

TRANSIENT RECEPTOR POTENTIAL (TRP) channels are a very large family of integral membrane proteins, which form cationic channels with diverse profiles with regard to their mechanisms of activation, electrophysiological and biophysical properties, and physiological functions (3, 4, 22, 28). On the basis of structural relatedness, mammalian TRP proteins have been categorized into six subfamilies: TRPC (canonical), TRPV (vanilloid receptor related), TRPM (melastatin related), TRPP (polycystin related), TRPML (mucolipin-like), and TRPA (ankyrin) (3, 4, 9, 21, 22). TRPM2, formerly known as TRPC7 (24) or LTRPC2 (27), forms Ca2+-permeable cationic channels that are specifically activated by intracellular adenosine diphosphoribose (ADPR) (27, 32). Numerous factors, including NAD, H2O2, cADPR, and intracellular Ca2+, have been reported to activate TRPM2 channels independently of or synergistically with ADPR (8, 10, 17, 20, 32, 34). TRPM2 mRNA is highly expressed in the brain and is also detected in many other tissues, including the heart, placenta, lung, kidney, spleen, and ovary (27, 33). TRPM2 or TRPM2-like functional responses have been documented in striatal neurons (12), U973 monocytes and Jurkat cells (27, 32), neutrophils (10), insulinoma cells (8, 13), and microglia (18). ADPR and other endogenous activators or synergizing ligands, and TRPM2 channels are thought to represent an intrinsic mechanism providing for Ca2+ entry into cells and regulating cellular functions (32), or causing cell death, by disrupting cellular homeostasis in response to changes in cellular redox states (8).

TRPM2 proteins comprise an ion channel core domain and cytoplasmic NH2 and COOH termini (Fig. 1). Whereas no specific functional roles have been designated for the NH2 terminus [residues 1–761 in human TRPM2 (hTRPM2)], a major distal part (residues 1236–1503, termed the NUTD9-H domain) of the COOH terminus, which encompasses residues 1049–1503, is homologous to the NUTD9 proteins, ADPR pyrophosphatases belonging to the nudix hydrolase family, and containing the binding site for ADPR (19, 27). The ion channel domain (residues 762–1048) is composed of six transmembrane (6TM) segments (S1–S6) and a pore loop between S5 and S6, similar to the voltage-gated potassium channels and other 6TM channels, including cyclic nucleotide-gated channels. Nevertheless, sequence analysis of these closely related channels, or even of the TRP channel family members, has led to the identification of sequence differences as well as sequence homology. The functional implications remain yet to be established. Sequence alignment of the pore regions of the TRP superfamily indicates that, unlike their close relatives in the TRPC and TRPV subfamilies, all the members of the TRPM subfamily contain a pair of cysteine residues (positions 996 and 1008 in the human TRPM2 subunit) of which the first is completely conserved within the TRPM subfamily and the second is present at slightly different positions (Fig. 1). In this study, we combined site-directed mutagenesis with biochemical and electrophysiological approaches to investigate the potential role of these conserved cysteine residues in TRPM2 channel function. Substitution of cysteine with either alanine or serine generated mutant channels that were functionally unresponsive to ADPR. Furthermore, biochemical experiments showed that loss of the channel function was not due to effects on protein expression, membrane trafficking, or localization. The mutant subunits also showed normal interaction with neighboring subunits. However, coexpression of wild-type with mutant subunits markedly reduced the ADPR-evoked currents. These results taken together suggest that cysteine residues in the pore region of TRPM2 play an obligatory role in channel function.


Figure 1
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  		Fig. 1. Schematic drawing of the membrane topology of a melastatin-related transient receptor potential protein (TRPM2) and sequence alignments of the pore regions of the TRPM subfamily. Top: membrane topology of the TRPM2 channel subunit. S1–S6 represent the six transmembrane segments and P represents the pore loop between S5 and S6. The TRP box indicates a stretch of relatively highly conserved amino acid residues in the TRP superfamily. The NUTD9-H domain in the COOH-terminus is homologous to the NUTD9 proteins, where the TRPM2 channel agonist adenosine diphosphoribose (ADPR) is thought to bind. Bottom: amino acid residue alignments (using ClustalW, http://www.ebi.ac.uk/clustalw/) of the pore regions and part of the S5 and S6 segments of human (h) TRPM1–8 subunits and the mouse (m) TRPM2 subunit. Residues are numbered according to the hTRPM2 subunit. Residues studied are indicated (cysteine residues at 996 and 1008 in bold and the glutamic acid residue at 994 underlined).

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals, constructs, cell culture, and transfection. All the chemicals and reagents were purchased from Sigma unless otherwise specified. The cDNA encoding hTRPM2 protein with an NH2-terminal FLAG (epitope) tag was a kind gift from A. M. Scharenberg (University of Washington, Seattle, WA). The plasmid encoding the hTRPM2 subunit with a COOH-terminal EE (epitope) tag was constructed as follows. The hTRPM2 and tag sequences were amplified by PCR using the following pair of primers: 5'-ATG GAG CCC TCA GCC CTG AGG AAA GC-3' (forward primer) and 5'-CTA TTC CAT TGG CAT GTA TTC GTA GTG AGC CCC GAA CTC AG-3' (reverse primer). The PCR amplicon was inserted into the vector pCR2.1 (Invitrogen), and the fragment between the HindIII and EcoRV sites was excised and subcloned into pcDNA 3.1/myc-His vector (Invitrogen) between HindIII and PmeI sites. The coding sequences were verified by sequencing. Both FLAG-tagged and EE-tagged constructs were used in this study, and no noticeable differences were seen in their functional properties, and therefore, data, where appropriate, were pooled. Point mutations were introduced in the EE-tagged hTRPM2 construct using the QuikChange site-directed mutagenesis protocol (Stratagene) and were verified by sequencing.

Human embryonic kidney cells (HEK-293) were used in this study to express wild-type and mutant TRPM2 proteins. Cells were maintained in DMEM supplemented with 2 mM glutamine and 10% heat-inactivated FBS (all from Invitrogen) at 37°C under 5% CO2 humidified conditions. Transient transfection of HEK-293 cells with plasmids was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Electrophysiological recordings. Cells were grown in 35-mm petri dishes and transfected with 1 µg hTRPM2 plasmid [and 0.1 µg green fluorescent protein (GFP) plasmid]. Transfected cells were seeded on glass coverslips 12–24 h after transfection and used in the following 24–72 h. Whole-cell recordings were made at room temperature using an Axopatch 200B amplifier and analyzed using pCLAMP9 software (Axon Instruments). Patch electrodes with a resistance of 4–6 M{Omega} were fabricated from borosilicate glass capillaries (World Precision Instruments). Cells were held at –40 mV, and voltage ramps from –120 mV to 80 mV with 1-s duration were applied every 5 or 10 s. Extracellular solutions contained (in mM) 147 NaCl, 2 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 13 glucose (Na+ solution), or 149 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 13 glucose (K+ solution), or 13 glucose, or 110 CaCl2, 10 HEPES, and 13 glucose (Ca2+ solution). Intracellular solution contained 147 mM NaCl, 50 µM EDTA, 1 mM MgCl2, 10 mM HEPES, and 1 mM Na2-ATP. All of the solutions were maintained at pH 7.3 with NaOH and 300–315 mosM. ADPR stock solution (100 mM) was made in intracellular solution, kept at –20°C in aliquots, and diluted to the indicated concentrations in intracellular solution prior to use. Flufenamic acid (FFA) was freshly made as a 1 M stock solution in dimethyl sulfoxide (DMSO) and diluted in extracellular solution to a final concentration of 0.5 mM before application; there was no detectable effect of DMSO as previously reported (11). FFA was applied for 0.5–1 min via a rapid solution changer RSC-160 (Biologic Science Instruments, Grenoble, France). All of the data were presented as means ± SE, and statistical analysis was carried out using Student's t-test where appropriate.

Immunocytochemistry. Cells were prepared as described above for electrophysiological recordings without addition of GFP plasmid in the transfection. Immunocytochemistry was performed using protocols previously detailed (30). Briefly, we used mouse monoclonal anti-EE primary antibodies (1:1,000 dilution; Babco, Richmond, CA), and an FITC-conjugated anti-mouse IgG secondary antibody (1:200 dilution, Sigma).

Coimmunoprecipitation, biotinylation, and Western blot analysis. Cells were prepared in T25 flasks (~3 x 106 cells) and transfected with 3 µg plasmid as described above. For each flask, cells were collected into 1 ml of chilled PBS 24 h after transfection. For coimmunoprecipitation experiments, cells were pelleted at 4°C by centrifugation and were lysed at 4°C for 30 min in 200 µl of lysis buffer containing 50 mM Tris·HCl pH 8.0, 150 mM NaCl, 2 mM EGTA, 1% Triton X-100, 5% glycerol, and supplemented with a cocktail of protease inhibitors (Roche). After clearing by centrifugation at 4°C for 10 min, the supernatant was mixed with 20 µl pre-equilibrated Ezview red anti-FLAG M2 affinity gel beads (Sigma) and agitated at 4°C for 2 h. The beads were collected by centrifugation at 8,200 g for 30 s, kept on ice after the supernatant was removed, and washed three times, each time beads were resuspended in 1 ml of lysis buffer and centrifuged at 8,200 g for 30 s. After the final wash, the immunoprecipitated samples (beads) were resuspended in 40 µl of electrophoresis sample buffer containing 50 mM Tris·HCl, pH 6.8, 2% (wt/vol) SDS, 10% glycerol, 100 mM DTT, 0.05% bromophenol blue, boiled for 5 min, and centrifuged at 8,200 g for 30 s.

For biotin labeling experiments, HEK-293 cells were washed 3 times with PBS 24 h after transfection and incubated in 1 mg/ml sulfo-NHS-biotin (Pierce) for 30 min at 4°C. The cells were then washed three times in PBS containing 50 mM glycine, and lysed with lysis buffer (see above) for 30 min at 4°C. Biotin-conjugated cell surface proteins were purified with streptavidin-agarose. Affinity-purified protein complexes were denatured as described above for coimmunoprecipitation protein samples.

Western blot analysis was performed at room temperature. Protein samples were resolved on 8% SDS-PAGE gels and transferred to nitrocellulose membranes under semi-dry transfer conditions (Bio-Rad) for 1 h (constant current, 1 mA/cm2 membrane). After being blocked for 1 h, with 5% nonfat milk made in TBST solution (10 mM Tris·HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20), the membranes were incubated for 1 h with primary rabbit anti-EE antibody (1:2,000 dilution; Bethyl Laboratories, Montgomery, TX), and washed three times in TBST solution for 5 min each. The membranes were then incubated for 50 min with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:2,000 dilution; Santa Cruz Biotechnology), and washed three times in TBST solution as before. Proteins were visualized using SuperSignal West Pico chemiluminescent substrates, according to the manufacturer's protocol (Pierce), and images were captured on X-ray films.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Functional expression and properties of wild-type hTRPM2 channel. Whole-cell current recordings were carried out, following transient transfection of HEK-293 cells with TRPM2 cDNA, to confirm functional channel expression. Our preliminary experiments showed that the time course and the amplitude of the current responses were ADPR concentration-dependent, which is consistent with previous reports (17, 27). Thus a supermaximal concentration of ADPR (1 mM) was used throughout this study to obtain maximal functional responses. Figure 2A shows the current responses of a cell expressing wild-type TRPM2 channels to voltage ramps from –120 mV to 80 mV (left), and plots the current amplitudes at membrane potentials of –80 mV and +80 mV (right). Upon breaking into whole-cell configuration, large currents appeared, which reached a maximal level within 10 s. Currents exhibited typical linear current-voltage (I/V) relationships (Fig. 2B) with a reversible potential close to zero (–1.3 ± 0.2 mV, n = 5), indicating that they were elicited by nonselective cationic channels. The currents varied from ~0.5 nA to more than 10 nA, with a mean peak current amplitude of 5.0 ± 0.6 nA (n = 18) at a membrane potential of –80 mV. Such robust currents were never detected either in the TRPM2 channel-expressing cells, when recording using intracellular solution without ADPR (Fig. 2C, left), or in the nontransfected cells, using intracellular solution containing ADPR (Fig. 3B). To further confirm that the ADPR-evoked currents were mediated by the TRPM2 channels, we tested FFA, a TRPM2 channel antagonist (11), and also the sensitivity to calcium (20). As illustrated in Fig. 2B, FFA (0.5 mM) rapidly and almost completely inhibited the ADPR-evoked currents (Fig. 2C, middle). The inhibition was independent of the membrane potential and irreversible during washing for 5 min (Fig. 2B). Upon changing extracellular solution containing no calcium (replaced with an equal molar concentration of barium) to extracellular solution containing 2 mM calcium, the ADPR-evoked currents were remarkably increased (Fig. 2C, right). These results clearly indicate that the ADPR-evoked currents are the result of activation of functionally expressed TRPM2 channels and confirm that the electrophysiological and pharmacological properties observed here are similar to those reported by other groups (11, 17, 20, 27).


Figure 2
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  		Fig. 2. Functional expression and properties of hTRPM2 channels. A: representative current responses to voltage ramps from –120 mV to 80 mV of 1-s duration (left top) applied every 10 s in a cell expressing the TRPM2 channel (left bottom). Cells were held at –40 mV. The first three small current responses in gray, which are almost superimposed, were recorded in the cell-attached configuration. The large current responses in black were recorded after the whole-cell configuration was established. The current amplitudes at membrane potentials of –80 and 80 mV are plotted over time (right). The arrow indicates the first current recorded in the whole-cell configuration. Intracellular solution contained ADPR (1 mM). B: an example of the inhibition of ADPR-evoked currents by flufenamic acid (FFA; 0.5 mM for 1 min) at a membrane potential of –80 mV recorded every 5 s. Inset: current-voltage (I/V) relationship curves before (point 1: the ninth whole-cell recording, indicated with filled circle in Fig. 2B) and after FFA inhibition (point 2: the fifth recording after addition of FFA). The arrow indicates the first current recorded in the whole-cell configuration. Inhibition was almost complete and irreversible during washing for 5 min, and was also voltage-independent. C: summary of the mean peak currents evoked at –80 mV recorded from transfected cells with and without ADPR (1 mM) in the intracellular solution (left), before (–FFA), and after application of FFA (+FFA), from experiments shown in B (middle), and in extracellular solution containing 2 mM BaCl2 without CaCl2 (+Ba) and extracellular solution containing 2 mM CaCl2 (+Ca) (right). The numbers of cells examined are indicated above each bar. **P < 0.001 and ***P < 0.0001.

 

Figure 3
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  		Fig. 3. Mutations of cysteine residues result in loss of channel function. A: currents at a membrane potential of –80 mV from cells expressing TRPM2 wild-type (left) or C996S mutant channels (right) using voltage ramps as described in Fig. 2A. The arrow (right) indicates the first current recorded in the whole-cell configuration and the grey bars show the duration of application of FFA. B: summary of mean peak currents recorded from nontransfected human embryonic kidney-293 cells (HEK), and cells expressing epitope (EE)-tagged TRPM2 wild-type (WT), C996A, C996S, C1008A, or C1008S mutant channels. ***P < 0.0001, significant difference in the current amplitude of wild-type channels compared with mutant (C996A, C996S, C1008A, or C1008S) channels and nontransfected cells. C: Mean currents from cells expressing wild-type, C996S, or C1008S mutant channels in extracellular solutions containing 149 mM KCl or 110 mM CaCl2. **P < 0.005 indicates a significant difference in the current amplitude between wild-type and mutant channels (C996S or C1008S). The numbers of cells examined are indicated above each bar.

 
Effects of mutating C996 and C1008 on current responses to ADPR. To explore the potential functional role of cysteine residues at positions 996 and 1008 in the pore region, we first replaced these amino acid residues individually with alanine. In contrast to the robust ADPR-evoked currents observed following expression of the wild-type channels, cells expressing either C996A or C1008A mutant channels failed to respond to ADPR (Fig. 3B). We then introduced serine substitutions, because we reasoned that this manipulation would produce the smallest structural alteration, in that it changed an SH– for an OH– group. Surprisingly, ADPR also either failed to elicit currents or only evoked very small currents in cells expressing these mutant channels (Fig. 3, A and B).

Whole-cell currents were also recorded from cells expressing wild-type, C996S, or C1008S mutant channels in extracellular solutions containing K+ or Ca2+ as the major cations. In extracellular K+ and Ca2+ solutions, ADPR elicited substantial currents in cells expressing wild-type channels, but no current or very small currents in cells expressing C996S or C1008S mutant channels (Fig. 3C). To rule out the possibility that loss of channel function was due to a nonspecific mutational effect, we substituted alanine into a nonconserved glutamic acid residue (E994), one residue away from C996 (Fig. 1). Expression of the E994A mutant channels, like the wild-type channels, gave rise to robust ADPR-evoked currents (–6.1 ± 1.3 nA, n = 5), which were blocked by FFA (98.0 ± 1.3%, n = 5).

Effects on protein expression and membrane localization. The loss of channel function as a result of mutating these cysteine residues could be attributed to mutational defects in either protein expression or protein trafficking or both. To investigate these possibilities, we performed Western blot analysis, and immunostaining and biotin-labeling experiments. Fig. 4 shows the representative results. Clearly, there was no obvious difference in the expression levels of wild-type or mutant subunit proteins (C996A, C996S, C1008A, and C1008S) (Fig. 4A). Furthermore, immunoreactivity for wild-type, C996S, and C1008S mutant subunits was similar, showing dominant clustering around the plasma membrane (Fig. 4B). In addition, the biotin labeling experiments indicate that the levels of biotinylated TRPM2 protein at the plasma membrane were not greatly changed for wild-type and mutant subunits (Fig. 4C). Taken together, these results suggest that the mutation of C996 or C1008 did not significantly alter the protein expression, the trafficking of the protein, or the membrane localization, and therefore does not account for the loss of channel activity.


Figure 4
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  		Fig. 4. Loss of function for mutant TRPM2s is not due to defects in expression and membrane localization. A: Western blot analysis of whole-cell lysates from nontransfected HEK cells and HEK-293 cells expressing EE-tagged TRPM2 wild-type, C996A, C996S, C1008A, and C1008S mutant subunits using an anti-EE antibody. B: immunostaining of nontransfected HEK-293 cells, and HEK-293 cells expressing EE-tagged TRPM2 wild-type, C996S or C1008S mutant subunits using anti-EE antibody. Note that there was no detectable immunostaining in nontransfected HEK cells and that the immunoreactivity in cells expressing TRPM2 wild-type or mutant subunits was dominantly clustered around the plasma membrane. Furthermore, there was no significant difference in immunoreactivity between wild-type and mutant subunits. C: Western blot analysis of whole-cell lysates (top) and biotin-labeled membrane proteins (bottom) from cells expressing EE-tagged TRPM2 wild-type, C996S, and C1008S mutant subunits using anti-EE antibody. Similar results were observed in at least two independent experiments.

 
Coexpression of mutant subunits with wild-type subunit. TRPM2 channels are thought to form as tetramers, by analogy to voltage-gated potassium and other 6TM channels. The introduction of mutations in individual subunits could impair the subunit interactions which are required for the formation of functional channel complexes. To address this possibility, we coexpressed EE-tagged wild-type, C996S, or C1008S subunits with FLAG-tagged wild-type subunits, and performed coimmunoprecipitation. As shown in Fig. 5A, coexpression did not change the expression levels of either the EE-tagged wild-type, C996S, or C1008S mutant subunit proteins (top) or the FLAG-tagged wild-type subunit proteins (middle). The amount of EE-tagged wild-type, C998S, and C1008S mutant subunit proteins, pulled down with the coexpressed FLAG-tagged wild-type subunit proteins, using an anti-FLAG antibody, was similar (bottom). Similar results were also seen in reciprocal coimmunoprecipitation experiments using an anti-EE antibody (data not shown). Taken together, these results indicate that C996S and C1008S mutant subunits interact with neighboring wild-type subunits to the same extent as wild-type subunits interact with each other.


Figure 5
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  		Fig. 5. Mutant TRPM2 subunits when coexpressed with wild-type subunits show normal subunit interaction, but significantly reduced channel function. A: Western blot analysis of whole-cell lysates (top) using an anti-EE antibody. Western blot analysis using anti-FLAG antibody (middle) or using anti-EE antibody (bottom) of immunoprecipitated proteins pulled down using anti-FLAG antibody. Cells were transfected with plasmids for the pairs of wild-type or mutant subunits indicated above. Similar results were seen in two independent experiments. B: Current responses were recorded every 5 s from cells transfected with EE-tagged wild-type (top left), EE-tagged wild-type and C996S mutant subunits (top right), or EE-tagged wild-type and C1008S mutant subunits (bottom left). Insets: the I/V relationship curves before (point 1) and after (point 2) inhibition by FFA. The results from all the experiments are summarized as mean data (bottom right) and the numbers of cells examined are indicated above each bar. *P < 0.05, and ***P < 0.0001. A 1:1 plasmid ratio was used to coexpress wild-type and mutant subunits.

 
We next examined the functional effects of C996S and C1008S mutant subunits by recording the ADPR-evoked currents from cells expressing wild-type subunit alone, wild-type subunit coexpressed with C996S subunit, or wild-type subunit coexpressed with C1008S subunit. Figure 5 shows examples of current recordings. A significant reduction in ADPR-evoked current amplitude was observed when wild-type subunit was coexpressed with either C996S or C1008S mutant subunit. The mean amplitude of ADPR-evoked peak currents was 0.3 ± 0.06 nA (n = 8) from cells coexpressing wild-type and C996S subunits, 1.2 ± 0.2 nA (n = 11) from cells coexpressing wild-type and C1008S subunits and 4.7 ± 0.5 nA (n = 13) from cells expressing wild-type subunit alone (Fig. 5). The average ADPR-evoked currents were reduced by 94% and 75% as a result of coexpression of wild-type subunit with C996S mutant subunit, and coexpression of wild-type subunit with C1008S mutant subunit, respectively.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main finding from this study, which combines site-directed mutagenesis with biochemical and electrophysiological approaches, is that the conserved cysteine residues in the pore region are obligatory for normal TRPM2 channel function.

TRP channels, in terms of the membrane spanning or channel core domains (Fig. 1), are homologous to the voltage-gated potassium channels and other 6TM channels. However, careful amino acid sequence analysis of, for example, the pore regions of these closely related channels or even for the TRP channel family members, led to the identification of sequence differences as well as sequence homology. Our present understanding of the functional implications is still poor, but is starting to emerge. For example, the majority of TRPM channels (TRPM1–3, 6–8) are permeable to Ca2+, whereas the TRPM4 and TRPM5 channels are not. This striking difference in ion permeability was elegantly localized to a set of specific amino acid residues in the pore region by Nilius and colleagues (25). In the present study, we investigated two cysteine residues in the pore region; the first one is completely conserved in the TRPM subfamily and the second is present at slightly different positions (Fig. 1). Substitution of either of them with alanine led to complete loss of the channel function (Fig. 3). These positions were not able to tolerate any structural modifications, since substitution with serine, which only replaces the SH– group in the side chain with an OH– group, still blunted channel function (Fig. 3). In contrast to C996 and C1008, mutation of a nonconserved residue (E994) gave rise to normal functional channels, indicating that the loss of function for the cysteine mutants examined here is not simply due to nonspecific mutational effects.

TRPM2 are cationic channels that are permeable to Na+, K+ and Ca2+. Considering that C996 and C1008 are located in the pore region, mutations of pore residues could potentially alter the ion selectivity as has already been shown for the TRPM4 and TRPV6 channels (25). However, whole-cell recordings show that cells expressing C996S and C1008S subunits in extracellular solutions containing Na+, K+, or Ca2+ as the major ion species all failed to produce significant ADPR-evoked currents, in contrast to cells expressing wild-type subunit (Fig. 3, B and C). This largely rules out the possibility that loss of channel function was due to altered ion permeation properties of the channel.

Loss of ion channel function could, however, be attributed to changes in protein expression or protein trafficking, e.g., KATP channels (1, 26) and P2X receptors (2). Alternatively, it may also be due to changes in the ability of the subunits to interact to form multiple-subunit complexes, as previously shown for cyclic nucleotide-gated channels (36), Ca2+-activated potassium channels (16), and chloride channels (31). However, our Western blotting, immunostaining, and biotin labeling experiments show that the mutant TRPM2 protein expression levels and membrane localization were very similar to those of the wild-type subunit (Fig. 4). Furthermore, the coimmunoprecipitation experiments indicate that there were no detrimental effects on the ability of the subunits to interact as a result of mutating these cysteine residues (Fig. 5A). Whole-cell current recordings from cells coexpressing mutant and wild-type subunits demonstrate that coexpression of mutant subunits dramatically reduced the ADPR-evoked currents (Fig. 5B). These electrophysiological results not only indicate that the wild-type and mutant subunits do interact to form "heteromeric" channels, but also provide further functional evidence to support the importance of these cysteine residues in TRPM2 channel function.

TRPM2 channels, like any other ligand-gated ion channels, in the simplest model are activated in two major steps, that is, agonist binding and channel gating (conformational changes elicited by agonist binding that lead to channel opening). For TRPM2 channels, the agonist ADPR has been shown to bind to the intracellular COOH-terminal NUDT9-H domain (19, 27), which is physically distant from the pore region (Fig. 1). Thus it is unlikely that these cysteine residues contribute to ADPR binding. One possibility is that they may be involved in the conformational changes that occur during channel gating. However, because the cysteine SH– group is essential for channel function, an alternative interpretation is that these residues could form disulfide bonds to maintain the tertiary structure and/or conformation of the channel complex. Experiments showed that ADPR-evoked currents were not affected by application of DTT, a reducing reagent, or 2-(trimethylammonium)ethylmethanethiosulfonate, a cysteine-specific modifying reagent (data not shown). Similar observations using these modifying reagents have previously been seen for instance in the ATP-gated P2X receptors, which contain ten conserved cysteine residues on the extracellular domain (14, 15). However, subsequent studies (5, 7) using site-directed mutagenesis revealed that these conserved cysteines in the P2X receptor are structurally important in maintaining receptor function. Further studies are required to determine whether these cysteine residues play a role in the channel gating and/or they contribute to the structural stability of the channel.

Coexpression of TRPM2 wild-type and cysteine mutant subunits resulted in a significant suppression of the ADPR-evoked currents, and in particular the effect resulting from coexpression with C996S mutant subunit is striking. The difference in the functional effects seen between C996S and C1008S mutant subunits might be because C996 is completely conserved and may occupy a position that is structurally and/or functionally more critical than C1008. This study demonstrates that mutant subunits exhibit normal protein expression, membrane trafficking, and subunit interactions; thus it is reasonable to assume that incorporation of wild-type and mutant subunits into tetrameric channels is binomial. In such a scenario, the suppression of channel function observed with coexpression of wild-type and C996S mutant subunits suggests that channels containing one or more C996S subunits are not functional. TRPM2 mRNA is abundantly expressed in the brain. Functional TRPM2 or TRPM2-like channels have also been observed in neurons and many other cell types (see introduction). A major obstacle in studying the physiological functions of TRPM2 channels is the lack of specific antagonists. The strong functional suppression by the C996S mutant subunit, regardless of the underlying mechanisms, could be feasibly explored as a useful tool to probe the physiological roles of TRPM2 channels in the native cellular milieu, as has been demonstrated in many previous studies (e.g., 6, 23, 29, 34, 35).

In summary, this study has combined site-directed mutagenesis, biochemical, and electrophysiological approaches to provide evidence to support that two cysteine residues in the pore region are obligatory for normal TRPM2 channel function. Both cysteine residues are present in all the other TRPM subfamily members and in particular C996 is completely conserved, and thus the present findings contribute to our evolving understanding of the structure and function of the TRPM channels.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Wellcome Trust and the Royal Society (to L.-H. Jiang).


   ACKNOWLEDGMENTS
 
We are grateful to Dr. A. M. Scharenberg for providing the hTRPM2 cDNA clone. We thank Prof. D. J. Beech for support and Dr. C. J. Milligan for critical comments on the manuscript.

Present address for H.-J. Mao: Shanghai Institute of Microsystems and Information Technology, Chinese Academy of Sciences, Shanghai 200050, People's Republic of China.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L.-H. Jiang, Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK (e-mail: l.h.jiang{at}leeds.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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