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J. Biol. Chem., Vol. 280, Issue 23, 22540-22548, June 10, 2005

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Alternative Splicing Switches the Divalent Cation Selectivity of TRPM3 Channels^*

Johannes Oberwinkler, Annette Lis, Klaus M. Giehl, Veit Flockerzi, and Stephan E. Philipp¶

From the Institut für Experimentelle und Klinische Pharmakologie und Toxikologie and Anatomisches Institut, Universität des Saarlandes, D-66421, Homburg, Germany

Received for publication, March 21, 2005 , and in revised form, April 8, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
TRPM3 is a poorly understood member of the large family of transient receptor potential (TRP) ion channels. Here we describe five novel splice variants of TRPM3, TRPM31–5. These variants are characterized by a previously unknown amino terminus of 61 residues. The differences between the five variants arise through splice events at three different sites. One of these splice sites might be located in the pore region of the channel as indicated by sequence alignment with other, better-characterized TRP channels. We selected two splice variants, TRPM31 and TRPM32, that differ only in this presumed pore region and analyzed their biophysical characteristics after heterologous expression in human embryonic kidney 293 cells. TRPM31 as well as TRPM32 induced a novel, outwardly rectifying cationic conductance that was tightly regulated by intracellular Mg²⁺. However, these two variants are highly different in their ionic selectivity. Whereas TRPM31-encoded channels are poorly permeable for divalent cations, TRPM32-encoded channels are well permeated by Ca²⁺ and Mg²⁺. Additionally, we found that currents through TRPM32 are blocked by extracellular monovalent cations, whereas currents through TRPM31 are not. These differences unambiguously show that TRPM3 proteins constitute a pore-forming channel subunit and localize the position of the ion-conducting pore within the TRPM3 protein. Although the ionic selectivity of ion channels has traditionally been regarded as rather constant for a given channel-encoding gene, our results show that alternative splicing can be a mechanism to produce channels with very different selectivity profiles.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The transient receptor potential (TRP)¹ gene family comprises at least 28 mammalian genes divided into seven subfamilies (1, 2). Most of the encoded proteins exhibit common structural features such as six predicted transmembrane (TM) domains with a putative pore loop between TM5 and TM6 and the so-called TRP box after TM6 (1, 2). Although all members of this group have been reported to form cationic channels, their mechanisms of activation, their regulation, and their biological functions are remarkably diverse. They also display a large variety of different cation selectivities (1, 2). For example, TRPM4 and TRPM5 have been described as impermeable for divalent cations (3–5), whereas TRPV5 and TRPV6 appear to be exclusively permeable for Ca²⁺ (6, 7). The diversity of TRP channels is further increased by the fact that most members of the TRP gene family can give rise to several different transcripts due to alternative splicing (8). In a few cases, the functional consequences of these alternative splice events are now beginning to emerge. For example, miss-plicing of TRPM6 transcripts is associated with a hereditary disorder called hypomagnesemia with secondary hypocalcemia (9, 10), and an amino-terminal-truncated variant of TRPM4 appears to modulate Ca²⁺ oscillations after receptor stimulation in T lymphocytes (11).

However, up to now, the largest number of different splice variants for any TRP family member has been described for TRPM3 (12, 13). Lee et al. (12) reported six splice variants of human TRPM3, which they named TRPM3a–f. Their lengths range from 1544 to 1579 amino acid residues. Functional data were only reported for the TRPM3a splice variant. Heterologously expressed TRPM3a exhibits a constitutive, Ca²⁺ concentration-dependent Ca²⁺ entry that can be augmented by Ca²⁺ store depletion or by stimulation of muscarinic receptors (12). In addition, Grimm et al. (13) reported one further human variant that has a total of only 1325 residues because of its considerably shorter carboxyl terminus (13). On its amino terminal end, however, this variant, which we will refer to as TRPM3₁₃₂₅ throughout this report, possesses an additional 153 amino acids completely missing in the other reported TRPM3 variants. As is the case with TRPM3a, TRPM3₁₃₂₅ generates constitutively active, Ca²⁺-permeable channels when heterologously expressed in human embryonic kidney 293 (HEK-293) cells. The activity of TRPM3₁₃₂₅ channels can reportedly be enhanced by hypo-osmotically induced cell swelling (13) and by D-erythro-sphingosine (14).

In this report we describe five novel TRPM3 splice variants that we cloned as full-length cDNA constructs from mouse brain. These variants are 1699–1721 amino acid residues long and are characterized by a novel amino terminal sequence of 61 residues not described so far. Interestingly, two of the novel variants, designated TRPM31 and TRPM2, differ only in a region between the fifth and sixth transmembrane domains where the pore-forming region of the channel is assumed. This region is 12 amino acids longer in TRPM31 compared with TRPM32; additionally, a proline is substituted by an alanine. The principal finding of our study is that this alteration of the primary structure induces a large change in the ionic selectivity of the resulting ion channels. We thus identify alternative splicing as a mechanism to modify the selectivity of TRPM3 channels.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cloning of TRPM3 Variants—Oligonucleotide primers A, 5'-GAG AGC TGA GCG CAG GCT G-3', and Z, 5'-TCC TGC AAC ACA CGG TAA GCC-3', were used to amplify Trpm3 transcripts after oligo(dT)₁₈ primed reverse transcription of mouse brain total RNA. For amplification we used the Long Expand PCR kit (Roche Applied Science) and the following conditions: addition of the enzymes at 62 °C followed by 2 min at 94 °C, 10 cycles (94 °C, 10 s; 62 °C, 30 s; 68 °C, 5 min), 25 cycles (94 °C, 10 s; 62 °C, 30 s; 68 °C, 5 min + 20 s/cycle) and 7 min at 68 °C. Sequencing both strands of subcloned fragments identified five independent clones encoding TRPM31, three encoding TRPM32, and three encoding TRPM33. Regarding TRPM34 and TRPM35, one clone was found for each splice variant (Fig. 1C). We introduced the ribosome binding site ACC GCC ACC and a Myc tag in-frame immediately 5' to the start codon of Trpm31 and Trpm32 cDNAs, which were subsequently cloned into pCAGGS-IRES-GFP (15) for transient dicistronic expression of TRPM3 together with the green fluorescent protein (GFP). To generate stably transfected clones the TRPM31 and TRPM32 cDNAs were fused in-frame to the enhanced yellow fluorescent protein (EYFP) cDNA in the pEYFP-N1 vector (BD Biosciences Clontech) allowing expression of TRPM3-EYFP fusion proteins. The sequences of Trpm31, Trpm32, Trpm33, Trpm34, and Trpm35 have been submitted to GenBank^TM under accession numbers AJ544532 [GenBank] , AJ544534 [GenBank] , AJ544533 [GenBank] AJ544535, and AJ544536 [GenBank] .

Cell Culture, Transfection, and Generation of Stable Cell Lines— HEK-293 cells were grown in minimal essential medium supplemented with 10% fetal calf serum in a humidified atmosphere containing 5% CO₂. Cells were passaged 3 times a week until they reached passage 30. For transient transfection, cells grown to 70–80% confluence were transfected with TRPM31, Myc-tagged TRPM31, Myc-tagged TRPM32, or pCAGGS-IRES-GFP (mock) using PolyFect (Qiagen, Hilden, Germany). Cells were either used directly or passaged 24 h after transfection to reduce cell density. For generation of clones stably expressing Myc-tagged TRPM31-EYFP or TRPM32-EYFP fusion proteins, transfected cells were kept for 5 weeks in 500 µg/ml G418. Single surviving cells were isolated by cell sorting using a MoFlo fluorescent-associated cell sorter (DakoCytomation, Hamburg, Germany) and expanded. Cell clones were tested for their EYFP fluorescence and for expression of TRPM3 in Western blots using monoclonal anti-Myc antibodies and anti-GFP antibodies (Roche Applied Science).

Detection of TRPM3 mRNA and Proteins—The 3450- and 1684-bp fragments, covering the entire coding sequence of the mouse Trpm31 cDNA, were labeled by random priming with [³²P]dCTP and served as probes for Northern blots. Filters were exposed to x-ray films with intensifying screens at -80 °C for 2 weeks before they were stripped and hybridized with a 239-bp cDNA fragment of the human glyceraldehyde-3-phosphate dehydrogenase as control.

In situ hybridizations were performed on 20-µm cryosections of perfusion-fixed brain (16) and fresh brain from C57 Bl6/J x 129 SVJ or NMRI mice with similar results. Sections from fresh tissue were air dried for 30 min, fixed for 20 min in 4% paraformaldehyde solution, washed three times in phosphate-buffered saline, and finally dehydrated and defatted in 70 and 100% ethanol. Oligonucleotide probes 5'-A GTG GGA GTG CAT GCT ATT GAG AAC GGT GA-3' and 5'-TT TGA GGG CCC ATG TCT CGT ATT GTA CAG TTC CTC TAG TC-3' labeled with [³⁵S]dATP using terminal deoxynucleotide transferase revealed the same distribution pattern of signals. Control reactions with a 10-fold excess of unlabeled probe resulted in background grain densities. Specimens were exposed to x-ray films for 5 days and then to Kodak NTB2 photoemulsion for 3 weeks.

Western blots were performed as described (17). Cells from four 3-cm dishes grown to 60–70% confluence were lysed in 200 µl of Laemmli buffer and analyzed using monoclonal anti-Myc antibodies and anti-GFP antibodies.

Electrophysiological Recordings—Standard tight-seal, whole cell patch clamp recordings were performed with an EPC9 amplifier controlled by Pulse software (HEKA, Lambrecht, Germany). Electrodes had resistances of 2.5–5 M measured in standard bath solution and were coated with Sigmacote (Sigma). TRPM3-expressing cells were identified by their green fluorescence on an inverted microscope (Axiovert 135; Zeiss) and used 24–96 h after transfection. Cells were voltage-clamped at -15 mV, and the instantaneous current-voltage relationship was probed at 1-s intervals with fast (1 mV/ms) voltage ramps ranging from -115 mV to +85 mV. During recordings that lasted longer than 60 s, the interval between voltage ramps was often increased to 10 s. From the voltage ramps, the current at -80 mV and +80 mV and the reversal potential were obtained by offline analysis.

Pipette solutions contained (in mM) 100 CsAsp, 45 CsCl, 10 EGTA, 10 HEPES or 130 CsAsp, 10 NaCl, 5 K₄BAPTA (1,2-Bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid), 10 HEPES. When indicated, either 1 or 10 mM MgCl₂ was added to the pipette solution. The resulting free Mg²⁺ concentrations were calculated with MaxChelator (www.stanford.edu/~cpatton/maxc.html) and are indicated in the figures. pH was adjusted to 7.2 (CsOH and HCl) and osmolality to 305–315 mosmol/kg (adjusted with H₂0 or glucose). A liquid junction potential of 15 mV was corrected for.

Cells were maintained during the recordings at room temperature in Ringer solution. All extracellular solutions (Table I) were based on 10 mM HEPES; pH was adjusted to 7.2 (hydroxide of main cation and HCl) and osmolality to 315–335 mosmol/kg (glucose or H₂0). Solutions were applied for short duration by a custom-made local perfusion system whose outlet was placed at <200 µm from the recorded cell.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Identification and Cloning of TRPM3 Variants from Mouse Brain—Screening public databases we identified two partial human TRPM3 cDNA clones, AL136545 [GenBank] and XM_0136545 (Fig. 1C). Both clones derive from a single gene locus on human chromosome 9. The corresponding mouse gene shows a highly similar organization with 28 exons and spans more than 850 kb on mouse chromosome 19b (NCBI gene number 226025, Fig. 1A). Primers A and Z (Fig. 1A) were deduced from the mouse genomic sequence. Their sequences are located 5' and 3' to stop codons flanking an open reading frame with a predicted translation start that is also present in the human cDNA of clone AL136545 [GenBank] . Using this primer combination, 5.2-kbp cDNA fragments from mouse brain total RNA were amplified by reverse transcription PCR and subcloned (Fig. 1B). Similar products were amplified from mouse eye (data not shown). Each clone contained the complete coding sequence of mouse Trpm3. We identified five different splice variants, designated TRPM31, TRPM32, TRPM33, TRPM34, and TRPM35 (Fig. 1C). Their amino acid sequences comprise 1699 up to 1721 amino acids, which are encoded by exons 1, 3–7, and 9–28. The predicted exons 2 and 8 (NCBI gene number 226025) are not present in the cDNA of these TRPM3 variants from mouse, but 18 amino acid residues encoded by the corresponding exon 8 have been detected in the recently described human hTRPM3f variant (12). Exon 2 encodes a 59-amino acid sequence that corresponds to the amino terminal part of the human TRPM3₁₃₂₅ variant (13) but is absent in the a–f variants of hTRPM3. Exon 1 codes for a completely novel amino terminal sequence that is not present in any of the already described human TRPM3 variants. Among 13 independent mouse cDNA clones, we could not find a single one that contained exon 1 together with 2 (Fig. 1C), suggesting that these exons are expressed in a mutually exclusive fashion. This could be explained either by tightly regulated posttranscriptional processing or by expression of these variants from alternative promoters.

View larger version (57K): [in this window] [in a new window]   FIG. 1. Identification of TRPM3 variants from mouse brain. A, schematic diagram of the mouse Trpm3 gene, comprising 28 exons. Location of primers A and Z used to amplify the cDNA by reverse transcription PCR and stop codons in-frame to the translation start are indicated. B, reverse transcription PCR amplification of 5.2-kbp fragments using primers A and Z. C, schematic presentation of TRPM3 with transmembrane domains 1–6, coiled coil region (cc), and TRP homology domain (Trp). Novel mouse TRPM3 protein variants shown as thick black lines are compared with the human variants hTRPM3a–f (12) and hTRPM31325 (13). The numbers of amino acid residues of each variant are indicated in parentheses. D, putative pore regions of TRPM31 and TRPM32 compared with the corresponding mouse sequences of TRPM6 (accession number NP_700466 [GenBank] ), TRPM7 (accession number NP_067425 [GenBank] ), TRPV5 (accession number JC7795), and TRPV6 (accession number CAD62684 [GenBank] . The 12 additional amino acid residues present in TRPM31 are indicated. Identical residues are boxed in black, conserved in gray. An aspartate residue that determines Ca2+ permeation of the TRPV5/TRPV6 pore is marked by an asterisk. Residues proposed to build the selectivity filter of TRPV6 are underlined (29).

Expression Pattern of TRPM3—We subsequently analyzed the expression pattern of the mouse Trpm3 gene by Northern blot and by in situ hybridization (Fig. 2). We found Trpm3 expression in brain and eye with transcripts of 1.8, 2.6, 3.7, 5.8, 7.6, 9.4, >12, and >15 kb. (Fig. 2A). In agreement with previous results (13), we could not detect Trpm3 transcripts in mouse kidney by Northern blot (data not shown). In situ hybridization experiments showed Trpm3 transcripts in several regions of the mouse brain such as the dentate gyrus, the intermediate lateral septal nuclei, the indusium griseum, and the tenia tecta (Fig. 2, C and D). Strongest Trpm3 expression was found in the epithelial cells of choroid plexus (Fig. 2, B–D) where transcripts could readily be detected in 20 µg of total RNA (Fig. 2A).

TRPM31 and TRPM32 Form Cation Channels Regulated by Intracellular Mg²⁺—Alternative splicing within exon 24 of both the mouse and the human TRPM3 gene leads to the presence of 12 additional amino acid residues and the additional replacement of an alanine by a proline residue (Fig. 1D) (12). Interestingly, this domain, which is present in mTRPM31 and hTRPM3c (12) but absent in all other variants, lies between the presumed fifth and sixth transmembrane domains (Fig. 1C). In analogy to the topologically similar members of the TRPV subfamily (19), it is likely that this part of the protein contributes to the pore-forming region of the channel (Fig. 1D). The variants TRPM31 and TRPM32 differ only in this region (Fig. 1, C and D). We set out to test whether this change in the primary sequence altered the biophysical characteristics of the resulting ion channels. To this end, we expressed TRPM3 variants in HEK-293 cells, which do not express TRPM3 endogenously (Fig. 2A).

Starting with TRPM31, we noticed that transfected cells, but not control cells, exhibit a constitutively active, outwardly rectifying current. This current was already visible directly after establishing the whole cell configuration (Fig. 3, A and B). Replacing the extracellular cations by the impermeant cation N-methyl-D-glucamine (NMDG⁺) shifted the reversal potential from -9.5 ± 0.5 to -66.2 ± 0.8 mV (n = 5), indicating that the current was predominately carried by cations (Fig. 3A). When we used pipette solutions without Mg²⁺, TRPM31-induced currents increased strongly within the first minutes of whole cell recording (Fig. 3, B and C). Conversely, when we increased the free Mg²⁺ concentration in the recording pipette to 9 mM (Fig. 3, C and D), TRPM31-induced currents vanished rapidly. Using 0.9 mM free Mg²⁺ in the recording pipette, currents of intermediate size were observed (Fig. 3D). These data indicate that TRPM31 is regulated by physiological concentrations of free intracellular Mg²⁺, which typically is found to be in the order of 0.5–1 mM (20).

We next turned to TRPM32 and asked whether this variant also forms a functional channel. In extracellular Ringer solution, TRPM32 currents were small and increased only slightly during prolonged recording (Fig. 4A). However, when we again replaced all extracellular cations with NMDG⁺, we observed large outward currents, which disappeared as soon as standard conditions were re-established (Fig. 4, A and B). Such currents were not observed in control cells measured under identical conditions. The absence of detectable inward currents under these conditions indicates that, similar to TRPM31, the TRPM32-induced conductance is mainly permeable to cations. We then tested whether intracellular Mg²⁺ also regulated TRPM32 channels. Very similar to our results with TRPM31, we found large currents in the absence of intracellular Mg²⁺ but no detectable currents with 7 mM free Mg²⁺ in the pipette solution (Fig. 4C). Therefore, block by intracellular Mg²⁺ appears to be a common property of the two splice variants.

View larger version (106K): [in this window] [in a new window]   FIG. 2. Expression pattern of TRPM3. A, Northern blot analysis of 5–10 µg of poly(A)+ RNA or 20 µg of total RNA (choroid plexus, eye) from mouse tissues (left and middle panels) or HEK-293 cells (right panel) using the mouse Trpm31 cDNA as probe and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as control. B–D, in situ hybridization of Trpm3 transcripts in frontal sections of distinct forebrain areas. B, x-ray film of the forebrain with signals in the choroid plexus of the lateral (I/II) and third (III) ventricles. C and D, dark field microscopy of forebrain sections: CP, choroid plexus; IG, indusium griseum; LSI, intermediate part of lateral septal nuclei; TT, tecta tenia; DG, dentate gyrus; MHb, medial habenular nucleus. C, inset, bright field microscopy (hematoxylineosin stained) of the lateral ventricle with the ependym (EP) and CP (signals as black grains marked by arrowheads).

Despite these precautions, however, the formal possibility exists that the observed outwardly rectifying currents do not flow through TRPM3 proteins but result from up-regulation of endogenous TRPM7 channels. We found, however, that the currents in TRPM31-expressing cells exhibited a time-dependent facilitation upon depolarization to values higher than +60 mV (Fig. 3E, arrows), similar to those described for TRPM4, TRPM5, and TRPM8 (4, 5, 24). Channels encoded by TRPM7 or TRPM6 do not show such a voltage-dependent facilitation (21, 25). Also, when we exposed TRPM32-expressing cells to an extracellular solution containing only NMDG⁺ (to evoke the large outward currents), a voltage-dependent facilitation of the current could be observed (Fig. 4D, arrows). Therefore, TRPM31 and TRPM32 channels show a unique combination of biophysical properties that are not found in any other member of the TRPM channel family.

Splicing Changes the Ion Selectivity of TRPM3 Channels—To determine the ionic selectivity of the TRPM31 and TRPM32 splice variants, we measured TRPM3-dependent currents under bi-ionic conditions and analyzed the reversal potential (Fig. 5). Fig. 5B shows that the reversal potential is significantly more positive in cells expressing TRPM32 compared with TRPM31-expressing cells when Ca²⁺ and NMDG⁺ are the only cations present in the extracellular solution. Similar results were obtained with Mg²⁺ instead of Ca²⁺. An estimation of the relative permeability ratios utilizing the Goldman-Hodgkin-Katz formalism (26) showed that TRPM32 channels are at least 10 times more permeable for Ca²⁺ (Fig. 5C) and at least 100 times more permeable for Mg²⁺ than TRPM31 channels (Fig. 5D). This large difference in ion permeation properties establishes that the ion-conducting pore of TRPM3 channels is affected by the sequence differences between the two splice variants 1 and 2 (Fig. 1C). It therefore provides evidence that this region of the protein is part of the ion-conducting pore of TRPM3. Furthermore, this finding proves conclusively that the observed currents after overexpressing TRPM3 proteins are mediated by these proteins and are not the result of up-regulated, endogenous conductances.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Heterologous expression of TRPM31 induces outwardly rectifying cation currents inhibited by intracellular Mg2+. A, current-voltage relationship of a TRPM31-expressing cell in standard Ringer or NMDG+ solution within 60 s after establishing the whole cell patch clamp configuration. B, time course of current densities in Ringer solution at +80 mV (squares) and -80 mV (circles) of TRPM31-expressing (open symbols) and control (closed symbols) cells at the times indicated after rupture of the patch. Intracellular solution was Mg2+ free. Number of cells as indicated. TRPM31 current densities are statistically different from control cells (e.g. at t = 300 s, p < 0.01). C, time course of currents in two TRPM31-expressing cells at +80 mV and -80 mV with pipette solution containing either 0 (left panel) or 9 mM free Mg2+ (right panel). D, statistical analysis of experiments similar to panel C at +80 mV. E, current response of a TRPM31-expressing cell during voltage steps from -115 to +85 mV as indicated in the lower panel. Arrows indicate time-dependent facilitation at strong depolarization.

View larger version (24K): [in this window] [in a new window]   FIG. 4. TRPM32 mediates a Mg2+-inhibited cation conductance. A, currents at +80 mV of a TRPM32-expressing and a control cell in Ringer or NMDG+ solution. B, current-voltage relationships under conditions and at times indicated in panel A (arrows). C, current densities at +80 mV of TRPM32-expressing cells (extracellular 145 mM NMDG+ and 10 mM Mg2+) with pipette solution containing either 0 or 7 mM free Mg2+ (number of cells as indicated, p < 0.001). D, current response of a TRPM32-expressing cell during voltage steps from -115 to +85 mV as indicated in the lower panel. Extracellular solution was either Ringer (left panel) or contained only NMDG+ as cation (right panel). Arrows indicate time-dependent facilitation at strong depolarization.

Divalent Inward Currents through TRPM32—

TRPM3 Channel Variants Are Differentially Regulated by Extracellular Cations—Comparison of TRPM31 and TRPM32 outward currents obtained in extracellular Ringer solution and a solution containing NMDG⁺ as the only cation implied that outward currents through TRPM32 are inhibited by cations in the Ringer solution (Figs. 3A and 4B). We were intrigued by the fact that this block affected only outward currents through TRPM32 but not through TRPM31. We therefore investigated the nature of this inhibition using solutions that contained only one permeable cation in addition to NMDG⁺. We found that Ca²⁺ blocks outward currents through TRPM32 as well as through TRPM31 dose dependently (Figs. 5A and 7B). Similar results were obtained for Mg²⁺ (Fig. 7B). On the other hand, the monovalent cations Na⁺ and K⁺ quite strongly reduced TRPM32 outward currents, whereas neither of them affected currents through TRPM31 (Fig. 7, A and B).

TRPM32 thus is inhibited by all cations tested on the extracellular side. It is important to note, however, that at their respective physiological concentrations, none of the cations blocked outward currents through TRPM32 completely. Nevertheless, these results imply that TRPM3 channel activity is tightly regulated by the concentration of extracellular cations. While TRPM31 channels are only sensitive to divalent cations, TRPM32 channels are sensitive to all extracellular cations.

Expression of TRPM3 Variants Increases Resting Ca²⁺ Levels—We compared intracellular Ca²⁺ concentrations in untransfected control cells to HEK-293 cells stably expressing either TRPM31 or TRPM32 at similar levels (Fig. 8A). In Ringer solution containing 2 mM Ca²⁺, TRPM32-expressing cells showed significantly (p < 0.001) increased steady state Ca²⁺ levels of 145 ± 2.6 nM compared with TRPM31-expressing cells (114 ± 2.3 nM) and control cells (84 ± 1.6 nM) (Fig. 8B). When we exposed the cells to an extracellular solution devoid of Ca²⁺ (containing 2 mM EGTA), intracellular Ca²⁺ levels dropped to 70–80 nM, irrespective of the expression of TRPM3 channels (Fig. 8B). Upon re-addition of 2 mM extracellular Ca²⁺, TRPM32-expressing cells showed an overshooting rise of the intracellular Ca²⁺ concentration (Fig. 8B). Such an overshoot was seen neither in TRPM31-expressing cells nor in control cells (Fig. 8B). These data indicate that TRPM32-expressing cells possess an increased permeability for Ca²⁺ ions under physiological extracellular ionic conditions that likely causes the larger basal intracellular Ca²⁺ levels. On the other hand, TRPM31-expressing cells seem to have a reduced permeability for Ca²⁺ (compared with TRPM32-expressing cells) as witnessed by the reduced Ca²⁺ influx after extracellular Ca²⁺ re-addition and the lower basal intracellular Ca²⁺ concentration. These observations agree with our electrophysiological data that TRPM32 channels have a much larger divalent permeability compared with TRPM31 channels (Figs. 5 and 6). They also correspond well with previous observations that the human TRPM3a and TRPM3₁₃₂₅ variants, which both contain the same pore region as TRPM32, mediate Ca²⁺ entry under similar experimental conditions (12, 13).

View larger version (23K): [in this window] [in a new window]   FIG. 5. TRPM31 and TRPM32 display large differences in their relative permeability ratios for divalent cations. A, comparison of TRPM31 and TRPM32 currents at +80 mV and -80 mV in extracellular solutions containing indicated amounts of Ca2+. B, reversal potential during the experiment shown in panel A. C and D, statistical analysis of reversal potential measurements in experiments similar to that shown in panel B during the application of solutions containing the indicated concentration of Ca2+ (C) or Mg2+ (D) as the only permeable ion. Continuous thin lines show the expected reversal potential calculated from Goldman-Hodgkin-Katz theory (26) for the indicated relative permeability ratios. Each point represents the mean of 3–15 independent measurements (at a divalent concentration of 10 mM p < 0.001, otherwise at least p < 0.05).

View larger version (27K): [in this window] [in a new window]   FIG. 6. Divalent cation currents through TRPM3 channels. A, currents at +80 mV and -80 mV through TRPM32 in extracellular isotonic Ca2+ solution (120 mM Ca2+). B, current voltage relationships at the times indicated in panel A. C, statistical analysis of peak current densities at -80 mV obtained in experiments similar to those shown in panel A. The current densities measured in TRPM32-expressing cells are significantly larger than in control or TRPM31-expressing cells at 60 and 120 mM extracellular Ca2+ (60 mM: p < 0.05; 120 mM: p < 0.001, n = 6–10 in each group). D, same as panel C but with extracellular Mg2+ as charge carrier. The current densities measured in TRPM32-expressing cells are significantly larger than in control or TRPM31-expressing cells at 60 and 120 mM extracellular Mg2+ (60 mM: p < 0.01; 120 mM: p < 0.05, n = 3–6 in each group).

View larger version (26K): [in this window] [in a new window]   FIG. 7. Inhibition of TRPM3-dependent currents by extracellular cations. A, comparison of TRPM31 and TRPM32 currents at +80 mV and -80 mV in extracellular solutions containing indicated amounts of Na+. Outward currents through TRPM31 are unaffected by extracellular Na+, whereas outward currents through TRPM32 are inhibited in a dose-dependent manner by these ions. B, statistical analysis of recordings similar to those shown in panel A and in Fig. 5A, with varying concentrations of Na+, K+, Ca2+, and Mg2+. Currents at +80 mV were normalized to currents measured in standard bath solution (arrows in panel A and in Fig. 5A, n = 3–15 for each data point). The solutions containing only monovalent cations were supplemented with 1 mM Mg2+ to minimize the influence of the endogenous MagNuM/MIC current. Additionally, the solutions used to measure block by extracellular K+ ions contained 2 mM Cs+ (see Table I) in order to block endogenous K+-selective channels.

View larger version (29K): [in this window] [in a new window]   FIG. 8. Calcium entry in TRPM3-expressing cells under physiological ionic conditions. A, Western blot of cells stably expressing Myc-tagged TRPM31-EYFP or Myc-tagged TRPM32-EYFP fusion proteins using anti-Myc (left panel) or anti-GFP antibodies, which also recognize EYFP. B, free intracellular Ca2+ concentration measured with fura-2 in cells maintained in Ringer solution (containing 2 mM Ca2+) or Ca2+-free solution (containing 2 mM EGTA). Each trace represents the mean of 106–182 individual cells from 2–4 independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Alternative Splicing Switches the Ion Selectivity of TRPM3 Channels—The selectivity of ion channels is thought to be determined by the geometry and charge distribution of the selectivity filter, usually envisioned as the narrowest part of the channel pore (26). Typically, all members of an ion channel family, such as voltage-gated Na⁺,K⁺,orCa²⁺ channels, share common ionic selectivities. The TRP family of ion channels is already somewhat unusual in this respect as it encompasses members with quite diverging cationic selectivity profiles (reviewed in Ref. 1). The Trpm3 gene adds extra complexity to this picture, because two channels can be expressed from this gene with entirely different ionic selectivities. One channel, TRPM31, preferentially conducts monovalent cation influx, whereas TRPM32 strongly favors divalent entry. In vivo, such a change in ionic selectivity must be expected to have considerable consequences for the function of the channel and the physiology of the cell that expresses it.

Previously, it has been shown that subtle manipulations of key amino acid residues can substantially influence the divalent selectivity of ion channels. Voltage-gated Na⁺ channels that are transformed to conduct Ca²⁺ ions by exchanging a lysine and/or an alanine to a glutamate residue are an extreme example (27). However, these changes in the primary amino acid sequence have been introduced artificially, whereas the variations in selectivity we describe for TRPM3 have evolved naturally and occur in vivo. Although alternative splicing is increasingly recognized as a potent mechanism to dynamically modify ion channel properties (28), it has not yet been implicated in directly modifying the selectivity of a channel. Interestingly, Trpm1, the closest relative of Trpm3, also encodes splice variants that differ in the pore-forming region (8) and might therefore represent a further example for such a mechanism.

Editing of RNA is another mechanism of posttranscriptional modification occurring in vivo. Deamidation of a selected adenosine in primary transcripts of non-NMDA ionotropic glutamate receptors leads to the substitution of a glutamine by an arginine within the channel pore. This has been shown to reduce the Ca²⁺ selectivity of those channels with important consequences for their physiology (27).

Locating the Ion-conducting Pore in TRPM Channels—The switch of ionic selectivity in TRPM3 variants is brought about by removing a short stretch of 12 amino acid residues and exchanging 1 further residue within the linker domain between the presumed fifth and sixth transmembrane regions (Fig. 1B). The differences in ion selectivity seen for the TRPM3 splice variants strongly indicate that this linker domain constitutes the pore of TRPM3. Although this domain could already be suspected to be the ion-conducting pore, due to direct evidence obtained for TRPV1, TRPV4, TRPV5, and TRPV6 channels (19), this prediction has not been confirmed up to now for any member of the TRPM subfamily.

Compared with the presumed pore regions of other members of the TRP family, the pore loop of TRPM3 is considerably longer by 8 (TRPM32) and 20 (TRPM31) additional amino acid residues (Fig. 1C). The domains that build the proposed selectivity filter of the Ca²⁺-selective TRPV5/V6 channels (29) are conserved in TRPM3 proteins. The splicing within the TRPM3 channel pore introduces additional, positively charged amino acid residues into this domain. This might decrease the Ca²⁺ permeability of TRPM31 compared with TRPM32, perhaps simply because of increased electrostatic repulsion. In line with this reasoning, in AMPA and kainate receptors the afore-mentioned replacement of a glutamine by a positively charged arginine residue by RNA editing reduces the Ca²⁺ selectivity of those channels as well (30). Conversely, artificially introducing negatively charged glutamate residues in the pore of Na⁺ channels increases their divalent selectivity (27).

Block of TRPM3 Channels by Intra- and Extracellular Cations—Our data show that both TRPM31 and TRPM32 are regulated by physiological concentrations of intracellular Mg²⁺, similar to related members of the TRPM family such as TRPM6 and TRPM7 (21, 22). Previously, the short human variant TRPM3₁₃₂₅ has been reported to mediate increased calcium entry when the osmolality of the extracellular solution was shifted from 300 to 200 mosmol/kg (13), a stimulus that induces considerable swelling of the cells. Conceivably, the Mg²⁺ dependence that we observed in whole cell patch clamp experiments offers a mechanistic explanation of the sensitivity to changes in osmolality. Because the intracellular cation concentration and the cell volume are mutually and inversely related, regulation of TRPM3 activity by intracellular Mg²⁺ might explain why TRPM3 shows increased or reduced activity in hypotonic and hypertonic solutions, respectively.

Our experiments were performed in isotonic solutions. Under these conditions we found that TRPM3 currents are also tightly regulated by extracellular cations. However, only TRPM32 channel activity was influenced by the extracellular concentration of monovalent cations, whereas both TRPM3 variants were inhibited by high extracellular divalent concentrations. Block by extracellular Na⁺ is a highly uncommon feature of ion channels, but not entirely unprecedented. Inward rectifier (31) and, especially, HERG (32) potassium channels have also been shown to be inhibited by extracellular Na⁺. Although the block of TRPM32 by extracellular Na⁺ concentrations in the physiological range seemed to be severe in electrophysiological recordings (Fig. 7), it does not appear to be complete. The intracellular Ca²⁺ concentration in TRPM32-overexpressing cells was elevated (Fig. 8), indicating that, also under physiological extracellular conditions, Ca²⁺ can enter the cell through TRPM32 channels at a rate too low to be detectable in electrophysiological recordings but measurable in Ca²⁺ imaging experiments.

Functional Role of TRPM3—We found that TRPM3 is strongly expressed in the choroid plexus (Fig. 2). This tissue in the ventricles of the brain is responsible for the formation and the regulation of cerebrospinal fluid. It has been demonstrated that the concentrations of ions such as Ca²⁺ in cerebrospinal fluid are carefully regulated and are independent of variations in the plasma concentrations of these ions (33). The high expression in the choroid plexus might indicate involvement of TRPM3 proteins in the production of cerebrospinal fluid or the regulation of its ionic composition (Fig. 2A). At present we do not know which of the various TRPM3 variants are present in the choroid plexus. However, the high selectivity for divalent cations of TRPM32 channels makes this variant a good candidate to play a role in the regulation of divalent cation concentration in cerebrospinal fluid.

	FOOTNOTES

 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AJ544532 [GenBank] , AJ544534 [GenBank] , AJ544533 [GenBank] , AJ544535 [GenBank] , and AJ544536 [GenBank] .

^* This work was supported by grants from the Deutsche Forschungsgemeinschaft (Emmy-Noether Programm (to J. O.), SFB 530 (to S. E. P.)) and from the Universität des Saarlandes (HOMFOR). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Experimentelle und Klinische Pharmakologie und Toxikologie, Gebäude 46, Universität des Saarlandes, 66421 Homburg, Germany. Tel.: 49-6841-1626152; Fax: 49-6841-1626402; E-mail: stephan.philipp{at}uniklinik-saarland.de.

¹ The abbreviations used are: TRP, transient receptor potential; TRPM, TRP melastatin subfamily; TRPV, TRP vanilloid receptor subfamily; GFP, green fluorescent protein; EYFP, enhanced yellow fluorescent protein; HEK-293, human embryonic kidney 293; MagNuM, magnesium nucleotide-regulated metal ion current; MIC, magnesium-inhibited cation current.

	ACKNOWLEDGMENTS

 
We thank Ute Soltek, Heidi Löhr, Britta Leiner, and Stefanie Johne for excellent technical assistance and Barbara Niemeyer, Markus Hoth, and Adolfo Cavalié for helpful discussions and critical reading of the manuscript.

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