BACKGROUND
transient receptor potential  proteins form a large gene family of ion channels characterized by distinct activation mechanisms and biophysical properties.
by sequence homology, members of the family fall into six subfamilies .
there is mounting evidence that trp channels are involved in thermosensation, mechanosensation, smell and taste.
a subset of trp channels, called 'thermo-trps' , have been found to be highly temperature dependent and are directly involved in heat and cold sensation in the peripheral nervous system  <cit> .
several trp channels are mechanosensitive or activated by hypotonic challenge   <cit> .
trpc <dig> is specifically expressed in the rodent sensory epithelium of the vomeronasal organ  where it plays a critical role in signaling processes triggered by pheromones  <cit> .
more recently, evidence was obtained for a critical role of trp channels in taste perception.
thus, pkd2l <dig>  and pkd1l <dig> are co-expressed in a subset of taste receptor cells  which are responsible for the detection of sour tastants  <cit> .
trpm <dig> was found to be essential for sweet, bitter and umami taste perception.
trpm <dig> is immunolocalized in a subset of trc,  <cit>  and two independently generated trpm <dig> knock-out mouse models display diminished sweet, bitter and umami perception  <cit> .
interestingly, the sensitivity of trpm <dig> to temperature was suggested to be the molecular mechanism underlying the psychophysical phenomenon of "thermal taste", i.e.
enhanced sweetness perception with increasing temperature  <cit> .
studies of the biophysical properties of trpm <dig> in heterologous expression systems by us and other groups revealed a monovalent-selective cation channel, directly gated by intracellular calcium, which rises upon stimulation of guanine nucleotide-binding regulatory protein -coupled receptors  linked to phospholipases type c .
furthermore, trpm <dig> is regulated by voltage and phosphatidylinositol bisphosphate   <cit> .
trpm <dig>  the closest homologue of trpm <dig>  displays a similar activation mechanism and channel properties  <cit> .
trpm <dig> and trpm <dig> have been proposed as molecular candidates for calcium-activated nonselective  cation channels observed in many excitable and non-excitable cells  <cit> .
can channels are assumed to be involved in membrane depolarization and, consequently, in the regulation of the intracellular calcium concentration.
mechanistically, however, the role of trpm <dig> in trc is poorly understood.
taste buds consist of four different cell types .
type ii taste receptor cells are assumed to be directly involved in sweet, bitter and umami perception  <cit> .
taste compounds stimulate gpcrs for sweet and amino acids   <cit>  and bitter   <cit>  resulting in activation of the g-protein gustuducin .
consequently, calcium is released from internal stores via activation of plcβ <dig> and subsequent inositol- <dig> ,5-trisphosphate  binding to ip <dig> receptor type iii .
elevated calcium levels activate trpm <dig> leading to depolarization of the plasma membrane due to influx of na+.
the molecular events following trpm <dig> activation are still under debate.
recently, gap junction hemichannels were proposed to be involved downstream of trpm <dig> activation in taste signal transduction  <cit> .
it is also unknown whether trpm <dig> is associated with t1r/t2r and gαgust as one functional unit due to sub-cellular compartmentalization, or whether trpm <dig> represents an independent signaling component localized in distinct and specialized subcellular compartments of trc.
it is assumed that the gastrointestinal and the respiratory tracts have the ability to analyze the composition of their luminal content in order to adequately respond to toxins and irritants.
thus, signaling molecules enriched in trc have also been identified in populations of solitary epithelial cells in organs other than taste buds.
for example, gαgust was found in brush cells of the gastrointestinal tract and in pancreatic duct cells  <cit> .
bitter taste receptors, t2rs, gαgust, plcβ <dig> and the ip3iii receptor were immunolocalized in solitary chemosensory cells  in the larynx and respiratory epithelium  <cit> .
gαgust was found to be expressed in l type enteroendocrine cells in the human colon  <cit> .
these observations support the notion that a population of specialized epithelial cells is present in different organs and may constitute part of a diffuse chemosensory system  <cit> .
at present, common functional, morphological and biochemical criteria defining this putative cell system or these systems are not available and are the subject of intensive investigation.
here we report that trpm <dig> is highly abundant in rodent chemosensory organs including taste buds, the olfactory epithelium and the vno, as well as in a subset of solitary cells distributed throughout the epithelia of the respiratory system and the gastrointestinal tract.
the latter cells display morphological and biochemical cell features of brush cells .
collectively, our data suggest that trpm <dig> expression defines a population of specialized chemosensory cells and, thus, may offer a new point of entry to dissect molecular mechanisms involved in the processing of environmental cues by the respiratory system and the gastrointestinal tract.
RESULTS
localization of trpm <dig> on the basolateral surface of taste receptor cells
to elucidate a biological role of trpm <dig> we set out to study its expression pattern in rodent tissues using an immunohistochemical approach.
to this end, we generated a polyclonal anti-trpm <dig> antibody  and tested its specificity in hek  <dig> cells transiently transfected with the cdna of mouse trpm <dig>  in western blots of whole-cell lysates obtained from hek  <dig> cells expressing either trpm <dig> or yfp-trpm <dig>  the anti-trpm <dig> antibody detected protein bands of expected sizes  .
the immunoreactivity was abolished by preincubation of the antibody with the corresponding immunization peptide.
next, we tested the ab- <dig> antibody by indirect immunofluorescence staining of hek  <dig> cells .
in contrast to untransfected cells, trpm5-expressing cells displayed a fluorescent signal, which could be completely blocked by the corresponding immunization peptide.
of note, the subcellular distribution of trpm <dig> immunoreactivity was similar to that observed by imaging of living hek  <dig> cells expressing trpm <dig> fused to a yfp tag .
to summarize, two independent approaches showed that the generated antibody ab- <dig> specifically recognized the mouse trpm <dig> protein.
to test whether the trpm5-specific antibody ab- <dig> is suitable for the detection of trpm <dig> in rodent tissues we performed immunohistochemistry of paraffin sections of valate papillae .
in line with recent reports  <cit> , ab- <dig> elicited a strong immunofluorescent signal in a subpopulation of trcs in taste buds, while the surrounding tongue tissue was negative for trpm <dig>  trpm <dig> immunoreactivity was blocked by the corresponding immunization peptide .
notably, we only detected a trpm <dig> immunofluorescent signal at the basolateral membrane of some trc.
in order to confirm this observation, we performed co-localization studies of trpm <dig> and the lectin ulex europaeus agglutinin , a membrane marker of all taste bud cells   <cit> .
in fact, we found that trpm <dig> is mainly localized at the basolateral cell surface of trc and is absent in the gustatory pore, readily stained by uea.
expression of trpm <dig> in chemosensory organs
due to its biophysical characteristics  trpm <dig> appears to be well suited to function in sensory cells.
therefore, we tested the hypothesis that trpm <dig> may be present in sensory organs other than taste buds.
while we did not detect trpm <dig> in the retina , we obtained positive signals in chemosensory organs: the olfactory epithelium and the vno .
in the main olfactory epithelium, we detected trpm <dig> in solitary epithelial cells whose nuclei were mainly localized superficially, at the level of the supporting cell layer .
occasionally, few nuclei of trpm <dig> immunopositive cells appeared in the basal part of the epithelium .
like typical ciliated olfactory cells, trpm5-expressing cells have elongated cell bodies that reach both the lumen and the basal membrane .
in addition, we detected trpm <dig> in sensory and non-sensory epithelia of the vno.
in the sensory epithelium, trpm <dig> was localized at the apical part of the cell , while in the non-sensory epithelium, trpm <dig> was present in solitary immunoreactive cells scattered throughout the luminal part of the epithelium .
thus, our data suggest that trpm <dig> might play an as yet unappreciated physiological role in olfaction of odorants and pheromones.
trpm <dig> is enriched in brush cells of the respiratory system
by means of rt-pcr and northern blot analysis, trpm <dig> expression has been demonstrated in many tissues  <cit> .
therefore, we systematically searched rodent organs for trpm <dig> protein expression by immunohistochemistry.
trpm <dig> could not be detected in liver, pancreas, kidney, spleen, lymph nodes, vessels, salivary glands, ovary, blood cells, fat cells, skeletal and smooth muscle .
we did, however, observe trpm <dig> immunoreactive cells in the respiratory system  as well as in the gastrointestinal tract .
in the respiratory system, trpm <dig> expression was restricted to solitary epithelial cells of the nose, trachea and bronchus .
occasionally, in some regions of the tracheal epithelium trpm5-expressing cells appeared in clusters .
remarkably, the apical surface of trpm5-positive cells contacted the lumen, while the basolateral part bore a long extension .
like in trcs, trpm <dig> in respiratory tract cells was restricted to the basolateral membrane .
the distribution and morphological characteristics of these trpm5-expressing cells were indicative of neuroendocrine cells, neuroendocrine bodies or brush cells  <cit> .
in order to assign the trpm5-expressing cells to one of these entities, we performed double immunostainings against trpm <dig> and cell-specific markers.
typical markers for neuroendocrine cells and bodies, i.e.
chromogranin a, protein gene product  <dig>  and calcitonin gene-related peptide  <cit>  were found not to co-localize with trpm <dig> .
it is well established that brush cells of the respiratory system and the gastrointestinal tract display unique morphological features not observed in any other epithelial cells  <cit> .
first, their numerous apical microvilli are highly enriched in filaments which extend basally into the supranuclear area.
second, brush cells contain microvilli with corresponding rootlets of filaments extending from the lateral cell surface.
consequently, fluorescence microscopy enables the detection of intense immunoreactivity of villin in the apical as well as in the lateral regions of brush cells  <cit> .
another remarkable feature of brush cells is that their enrichment in villin is invariably accompanied by an intense immunoreactivity to cytokeratin  <dig>  <cit> .
therefore, we used antibodies specific for villin or anti-cytokeratin  <dig> , for co-immunostainings of these proteins and trpm <dig> in tracheal tissue sections.
we noted an invariable co-localization of these protein markers and trpm <dig> in the same cells  indicating that trpm5-positive cells of the trachea are most probably brush cells.
rat alveolar epithelia contain solitary villin-positive cells considered to be brush cells  <cit> .
therefore, we tested whether these cells that we could detect with an anti-villin antibody, would also yield a positive trpm <dig> staining.
figure 3j–l shows that trpm <dig> and villin are invariably co-localized in solitary alveolar cells.
localization of trpm <dig> in the gastrointestinal tract
brush cells are known to be expressed not only in the respiratory system, but throughout the gastrointestinal tract, and northern blot analysis indicates a considerable enrichment of trpm <dig> mrna in the intestine  <cit> .
thus, we extended our study to a detailed immunohistochemical analysis of the gut.
throughout the gastrointestinal tract, we detected solitary trpm5-positive cells .
in the stomach, trpm5-enriched cells were readily seen in the principal part of the gastric glands, most abundantly in the cardiac region .
in the small intestine  and colon  scarce solitary trpm5-positive cells were scattered throughout the epithelial sheet with a conspicuous enrichment in the villar region in the ileum .
similar to their counterparts in the tracheal epithelium, the trpm5-positive cells in the gastrointestinal tract share morphological features with brush cells .
typically, these cells directly face the luminal surface, and long basal extensions reach the lamina propria .
unlike the surrounding gut epithelial cells, trpm5-positive cells display a conspicuous apical microvillar tuft reminiscent of brush cells .
moreover, like in trc and respiratory brush cells, trpm <dig> expression sites were confined to the basolateral cell surface.
in order to determine whether or not the cells detected were bona fide brush cells, we performed immunohistochemical co-stainings of trpm5-positive cells with the brush cell marker ck <dig> .
most of the trpm5-expressing cells displayed a clear co-localization with ck <dig>  however, we found a minor ck18-negative fraction , either due to limited sensitivity, or because these cells represent a distinct subtype.
conversely, all cells staining positively for ck <dig> clearly expressed trpm <dig>
to summarize, we found that trpm <dig> is expressed throughout the gastrointestinal tract, specifically in solitary epithelial cells, which can be classified as brush cells based on cell distribution, morphological criteria and the presence of a specific marker protein.
discussion
studies on trp channels have significantly extended our knowledge of the molecular events underlying the sensation of temperature, mechanical force, smell and taste.
thus, trpm <dig> was recently shown to be enriched in trc and is essential for the perception of sweet, bitter and umami taste compounds  <cit> .
however, it is well documented in the scientific literature that the expression pattern of trpm <dig> is not restricted to taste buds.
these findings prompted us to test the hypothesis that trpm <dig> may play a broader functional role in chemosensation than initially suggested by experiments with trpm5-deficient mice.
to this end, we raised and tested anti-trpm <dig> antibodies whose staining pattern of taste receptor cells is fully congruent with previous work by other researchers  <cit> .
however, performing a more detailed analysis, we observed in addition that the basolateral plasma membrane of trc is readily stained, whereas the apical part, the gustatory pore, is spared – a principle applying to all other trpm <dig> positive cells investigated with the notable exception of cells in the sensory epithelium of the vno.
these observations provide further insight into the function of trpm <dig> in trc.
most other trp channels that are involved in sensory signaling are eo ipso receptors of extra- or intracellular messengers and are found at the exact location of primary signal detection.
this notion holds true for both direct signal detectors like heat- or cold-sensing trps  <cit>  as well as for trpc channels that are activated by a receptor and g protein-dependent signaling cascade like trp and trpl in the insect rhabdomere  <cit> , the trpc <dig> channel expressed in the dendritic knob of rodent vno  <cit>  or trpc <dig> expressed in microvillar cells of the main olfactory epithelium  <cit> .
our data suggest that trpm <dig> is not directly involved in the process of taste recognition.
activation of trpm <dig> may depolarize the basolateral plasma membrane resulting in the modulation of other voltage-dependent ion channels and subsequent neurotransmitter release.
alternatively, the trpm5-mediated depolarization of the basolateral membrane could modulate the function of gap junctions, and, thus change the physiological status of neighboring cells  <cit> .
our studies with other sensory organs strongly indicate that in addition to taste perception trpm <dig> is also involved in the processing of olfactory stimuli.
accordingly, we identified trpm <dig> in two chemosensory organs, vno and olfactory epithelia.
we detected trpm <dig> in solitary cells within the non-sensory epithelium and apically in the sensory epithelium of the vno.
interestingly, another member of the trp gene family, the ca2+-permeable trpc <dig> is also present on the apical cell surface of the sensory epithelium of the vno  <cit> .
thus, it is imaginable that in conjunction with trpc <dig>  trpm <dig> is required for the detection of pheromones.
in this context it should be recalled that trpm <dig> cdna was isolated from the vno  <cit> .
furthermore, can channel activity with electrophysical properties similar to trpm <dig> or trpm <dig> was measured in freshly isolated vno neurons  <cit> .
preliminary findings of our group using an anti-trpm <dig> antibody illustrate that trpm <dig> is highly expressed in the sensory epithelium of the vno where it localizes to the apical region of sensory cells like trpm <dig> .
these findings raise the possibility that in the vno, trpm <dig> and trpm <dig> are co-expressed and co-localized and might therefore assemble to form heteromultimeric channels whose subcellular trafficking and functional properties could differ from either channel expressed alone in other cells.
in the olfactory epithelium, trpm <dig> was found in scattered cells.
the morphology and distribution of trpm5-expressing cells are reminiscent of microvillar cells which are thought to be involved in sensing of odorants  <cit> .
by means of immunohistochemical criteria, it was recently shown that microvillar cells can be classified into at least two subtypes, one of which was denoted brush cells  <cit>  that were also found to be localized in the non-sensory epithelium of the vno  <cit> .
the physiological relevance of trpm <dig> expression in the vno and the olfactory epithelium still remains to be clarified by a meticulous analysis of odorant and pheromone sensing in trpm5-deficient mice.
while this manuscript was under review, lin w. et al.
<cit>  reported expression data similar to ours for trpm <dig> in the main olfactory epithelium.
furthermore, these authors provided evidence for an involvement of trpm <dig> in the sensing of semiochemicals, compatible with our hypothesis that trpm <dig> may take part in chemosensation.
it was recently postulated that a population of specialized cells of the respiratory epithelium and gastrointestinal tract forming the so-called diffuse chemosensory system participates in the recognition and processing of diverse environmental cues  <cit> .
this assumption was recently substantiated by the immunolocalization of signaling proteins such as gαgust, t2r, plcβ <dig> and the ip3iii receptor in solitary intestinal epithelial cells and in cells of the respiratory system.)
<cit> .
in accordance with these earlier findings, bezençon et al.
took advantage of trpm <dig> promoter-based egfp transgenic mice and identified gfp-positive cells in the gastrointestinal tract which partially co-express a set of taste signaling proteins mentioned above  <cit> .
these observations are in line with our data demonstrating trpm <dig> expression in scattered epithelial cells of the respiratory system and gastrointestinal tract.
the cell body of trpm5-enriched cells is bottle-shaped.
the apical elongated narrow cell pole directly contacts the lumen and protrudes beyond the neighboring epithelial cells.
on the basolateral cell pole, trpm5-expressing cells have a long process contacting the lamina propria.
thus, the distribution and morphological characteristics of trpm5-immunopositive cells conspicuously share numerous features with brush cells  <cit> .
to identify the cellular origin of trpm5-immunopositive cells, we resorted to double immunofluorescence staining using antibodies directed against villin or ck <dig>  proteins known to be enriched in brush cells  <cit> .
we observed an invariable co-localization of these marker proteins with trpm <dig> in the respiratory system and a predominant co-localization in the duodenum.
brush cells were initially defined by morphological criteria, and a clear biological role of this cell population still remains elusive.
hypotheses about these cells' functional role range from absorptive processes  <cit> , mechanosensation  <cit> , secretion  <cit>  or a contribution to the local immunological defense barrier  <cit> .
in the respiratory system and the gastrointestinal tract, brush cells may play a role in sensation of chemical compounds in the lumen  <cit> .
the latter notion is derived from two salient observations: firstly, brush cells display morphological features similar to trc.
both cell types are characterized by an apical tuft of stiff microvilli with long rootlets into the cytoplasm lacking classical exocytotic vesicles or synapses  <cit> .
secondly, several signaling proteins pertinent to taste perception were identified in a subset of brush cells, including α-gustducin  <cit>  and no-synthase  <cit> .
to our knowledge, trpm <dig> is the first protein identified to be specifically expressed in brush cells of the gastrointestinal tract and the respiratory system.
these results lend further support to the view that taste cells and brush cells share a common functional principle, i.e.
chemosensation, and that brush cells are an important cellular correlate for chemosensation in the periphery.
CONCLUSIONS
the abundant and specific expression of trpm <dig> in taste receptor cells, the vno and the olfactory epithelium is consistent with the assumption that trpm <dig> has a common functional role in sensation of taste compounds, olfaction of odorants and pheromones.
trpm <dig> is the first protein identified to show high and invariable enrichment in brush cells of the gastrointestinal tract and the respiratory system.
our data are suggestive of brush cells as a specialized population of epithelial cells potentially involved in chemosensation and of trpm <dig> as a characteristic molecular constituent of the sensory machinery of these cells.

 transient receptor potential proteins form a large gene family of ion channels characterized by distinct activation mechanisms and biophysical properties. by sequence homology, members of the family fall into six subfamilies. there is mounting evidence that trp channels are involved in thermosensation, mechanosensation, smell and taste. a subset of trp channels, called'thermo-trps', have been found to be highly temperature dependent and are directly involved in heat and cold sensation in the peripheral nervous system. several trp channels are mechanosensitive or activated by hypotonic challenge. trpc is specifically expressed in the rodent sensory epithelium of the vomeronasal organ where it plays a critical role in signaling processes triggered by pheromones. more recently, evidence was obtained for a critical role of trp channels in taste perception. thus, pkd2l and pkd1l are co-expressed in a subset of taste receptor cells which are responsible for the detection of sour tastants. trpm was found to be essential for sweet, bitter and umami taste perception. trpm is immunolocalized in a subset of trc, and two independently generated trpm knock-out mouse models display diminished sweet, bitter and umami perception. interestingly, the sensitivity of trpm to temperature was suggested to be the molecular mechanism underlying the psychophysical phenomenon of "thermal taste", i.e. enhanced sweetness perception with increasing temperature. studies of the biophysical properties of trpm in heterologous expression systems by us and other groups revealed a monovalent-selective cation channel, directly gated by intracellular calcium, which rises upon stimulation of guanine nucleotide-binding regulatory protein -coupled receptors linked to phospholipases type c. furthermore, trpm is regulated by voltage and phosphatidylinositol bisphosphate. trpm the closest homologue of trpm displays a similar activation mechanism and channel properties. trpm and trpm have been proposed as molecular candidates for calcium-activated nonselective cation channels observed in many excitable and non-excitable cells. can channels are assumed to be involved in membrane depolarization and, consequently, in the regulation of the intracellular calcium concentration. mechanistically, however, the role of trpm in trc is poorly understood. taste buds consist of four different cell types. type ii taste receptor cells are assumed to be directly involved in sweet, bitter and umami perception. taste compounds stimulate gpcrs for sweet and amino acids and bitter resulting in activation of the g-protein gustuducin. consequently, calcium is released from internal stores via activation of plcβ and subsequent inositol-,5-trisphosphate binding to ip receptor type iii. elevated calcium levels activate trpm leading to depolarization of the plasma membrane due to influx of na+. the molecular events following trpm activation are still under debate. recently, gap junction hemichannels were proposed to be involved downstream of trpm activation in taste signal transduction. it is also unknown whether trpm is associated with t1r/t2r and gαgust as one functional unit due to sub-cellular compartmentalization, or whether trpm represents an independent signaling component localized in distinct and specialized subcellular compartments of trc. it is assumed that the gastrointestinal and the respiratory tracts have the ability to analyze the composition of their luminal content in order to adequately respond to toxins and irritants. thus, signaling molecules enriched in trc have also been identified in populations of solitary epithelial cells in organs other than taste buds. for example, gαgust was found in brush cells of the gastrointestinal tract and in pancreatic duct cells. bitter taste receptors, t2rs, gαgust, plcβ and the ip3iii receptor were immunolocalized in solitary chemosensory cells in the larynx and respiratory epithelium. gαgust was found to be expressed in l type enteroendocrine cells in the human colon. these observations support the notion that a population of specialized epithelial cells is present in different organs and may constitute part of a diffuse chemosensory system. at present, common functional, morphological and biochemical criteria defining this putative cell system or these systems are not available and are the subject of intensive investigation. here we report that trpm is highly abundant in rodent chemosensory organs including taste buds, the olfactory epithelium and the vno, as well as in a subset of solitary cells distributed throughout the epithelia of the respiratory system and the gastrointestinal tract. the latter cells display morphological and biochemical cell features of brush cells. collectively, our data suggest that trpm expression defines a population of specialized chemosensory cells and, thus, may offer a new point of entry to dissect molecular mechanisms involved in the processing of environmental cues by the respiratory system and the gastrointestinal tract.  localization of trpm on the basolateral surface of taste receptor cells to elucidate a biological role of trpm we set out to study its expression pattern in rodent tissues using an immunohistochemical approach. to this end, we generated a polyclonal anti-trpm antibody and tested its specificity in hek cells transiently transfected with the cdna of mouse trpm in western blots of whole-cell lysates obtained from hek cells expressing either trpm or yfp-trpm the anti-trpm antibody detected protein bands of expected sizes. the immunoreactivity was abolished by preincubation of the antibody with the corresponding immunization peptide. next, we tested the ab- antibody by indirect immunofluorescence staining of hek cells. in contrast to untransfected cells, trpm5-expressing cells displayed a fluorescent signal, which could be completely blocked by the corresponding immunization peptide. of note, the subcellular distribution of trpm immunoreactivity was similar to that observed by imaging of living hek cells expressing trpm fused to a yfp tag. to summarize, two independent approaches showed that the generated antibody ab- specifically recognized the mouse trpm protein. to test whether the trpm5-specific antibody ab- is suitable for the detection of trpm in rodent tissues we performed immunohistochemistry of paraffin sections of valate papillae. in line with recent reports, ab- elicited a strong immunofluorescent signal in a subpopulation of trcs in taste buds, while the surrounding tongue tissue was negative for trpm trpm immunoreactivity was blocked by the corresponding immunization peptide. notably, we only detected a trpm immunofluorescent signal at the basolateral membrane of some trc. in order to confirm this observation, we performed co-localization studies of trpm and the lectin ulex europaeus agglutinin, a membrane marker of all taste bud cells. in fact, we found that trpm is mainly localized at the basolateral cell surface of trc and is absent in the gustatory pore, readily stained by uea. expression of trpm in chemosensory organs due to its biophysical characteristics trpm appears to be well suited to function in sensory cells. therefore, we tested the hypothesis that trpm may be present in sensory organs other than taste buds. while we did not detect trpm in the retina, we obtained positive signals in chemosensory organs: the olfactory epithelium and the vno. in the main olfactory epithelium, we detected trpm in solitary epithelial cells whose nuclei were mainly localized superficially, at the level of the supporting cell layer. occasionally, few nuclei of trpm immunopositive cells appeared in the basal part of the epithelium. like typical ciliated olfactory cells, trpm5-expressing cells have elongated cell bodies that reach both the lumen and the basal membrane. in addition, we detected trpm in sensory and non-sensory epithelia of the vno. in the sensory epithelium, trpm was localized at the apical part of the cell, while in the non-sensory epithelium, trpm was present in solitary immunoreactive cells scattered throughout the luminal part of the epithelium. thus, our data suggest that trpm might play an as yet unappreciated physiological role in olfaction of odorants and pheromones. trpm is enriched in brush cells of the respiratory system by means of rt-pcr and northern blot analysis, trpm expression has been demonstrated in many tissues. therefore, we systematically searched rodent organs for trpm protein expression by immunohistochemistry. trpm could not be detected in liver, pancreas, kidney, spleen, lymph nodes, vessels, salivary glands, ovary, blood cells, fat cells, skeletal and smooth muscle. we did, however, observe trpm immunoreactive cells in the respiratory system as well as in the gastrointestinal tract. in the respiratory system, trpm expression was restricted to solitary epithelial cells of the nose, trachea and bronchus. occasionally, in some regions of the tracheal epithelium trpm5-expressing cells appeared in clusters. remarkably, the apical surface of trpm5-positive cells contacted the lumen, while the basolateral part bore a long extension. like in trcs, trpm in respiratory tract cells was restricted to the basolateral membrane. the distribution and morphological characteristics of these trpm5-expressing cells were indicative of neuroendocrine cells, neuroendocrine bodies or brush cells. in order to assign the trpm5-expressing cells to one of these entities, we performed double immunostainings against trpm and cell-specific markers. typical markers for neuroendocrine cells and bodies, i.e. chromogranin a, protein gene product and calcitonin gene-related peptide were found not to co-localize with trpm. it is well established that brush cells of the respiratory system and the gastrointestinal tract display unique morphological features not observed in any other epithelial cells. first, their numerous apical microvilli are highly enriched in filaments which extend basally into the supranuclear area. second, brush cells contain microvilli with corresponding rootlets of filaments extending from the lateral cell surface. consequently, fluorescence microscopy enables the detection of intense immunoreactivity of villin in the apical as well as in the lateral regions of brush cells. another remarkable feature of brush cells is that their enrichment in villin is invariably accompanied by an intense immunoreactivity to cytokeratin. therefore, we used antibodies specific for villin or anti-cytokeratin, for co-immunostainings of these proteins and trpm in tracheal tissue sections. we noted an invariable co-localization of these protein markers and trpm in the same cells indicating that trpm5-positive cells of the trachea are most probably brush cells. rat alveolar epithelia contain solitary villin-positive cells considered to be brush cells. therefore, we tested whether these cells that we could detect with an anti-villin antibody, would also yield a positive trpm staining. figure 3j–l shows that trpm and villin are invariably co-localized in solitary alveolar cells. localization of trpm in the gastrointestinal tract brush cells are known to be expressed not only in the respiratory system, but throughout the gastrointestinal tract, and northern blot analysis indicates a considerable enrichment of trpm mrna in the intestine. thus, we extended our study to a detailed immunohistochemical analysis of the gut. throughout the gastrointestinal tract, we detected solitary trpm5-positive cells. in the stomach, trpm5-enriched cells were readily seen in the principal part of the gastric glands, most abundantly in the cardiac region. in the small intestine and colon scarce solitary trpm5-positive cells were scattered throughout the epithelial sheet with a conspicuous enrichment in the villar region in the ileum. similar to their counterparts in the tracheal epithelium, the trpm5-positive cells in the gastrointestinal tract share morphological features with brush cells. typically, these cells directly face the luminal surface, and long basal extensions reach the lamina propria. unlike the surrounding gut epithelial cells, trpm5-positive cells display a conspicuous apical microvillar tuft reminiscent of brush cells. moreover, like in trc and respiratory brush cells, trpm expression sites were confined to the basolateral cell surface. in order to determine whether or not the cells detected were bona fide brush cells, we performed immunohistochemical co-stainings of trpm5-positive cells with the brush cell marker ck. most of the trpm5-expressing cells displayed a clear co-localization with ck however, we found a minor ck18-negative fraction, either due to limited sensitivity, or because these cells represent a distinct subtype. conversely, all cells staining positively for ck clearly expressed trpm to summarize, we found that trpm is expressed throughout the gastrointestinal tract, specifically in solitary epithelial cells, which can be classified as brush cells based on cell distribution, morphological criteria and the presence of a specific marker protein. discussion studies on trp channels have significantly extended our knowledge of the molecular events underlying the sensation of temperature, mechanical force, smell and taste. thus, trpm was recently shown to be enriched in trc and is essential for the perception of sweet, bitter and umami taste compounds. however, it is well documented in the scientific literature that the expression pattern of trpm is not restricted to taste buds. these findings prompted us to test the hypothesis that trpm may play a broader functional role in chemosensation than initially suggested by experiments with trpm5-deficient mice. to this end, we raised and tested anti-trpm antibodies whose staining pattern of taste receptor cells is fully congruent with previous work by other researchers. however, performing a more detailed analysis, we observed in addition that the basolateral plasma membrane of trc is readily stained, whereas the apical part, the gustatory pore, is spared – a principle applying to all other trpm positive cells investigated with the notable exception of cells in the sensory epithelium of the vno. these observations provide further insight into the function of trpm in trc. most other trp channels that are involved in sensory signaling are eo ipso receptors of extra- or intracellular messengers and are found at the exact location of primary signal detection. this notion holds true for both direct signal detectors like heat- or cold-sensing trps as well as for trpc channels that are activated by a receptor and g protein-dependent signaling cascade like trp and trpl in the insect rhabdomere, the trpc channel expressed in the dendritic knob of rodent vno or trpc expressed in microvillar cells of the main olfactory epithelium. our data suggest that trpm is not directly involved in the process of taste recognition. activation of trpm may depolarize the basolateral plasma membrane resulting in the modulation of other voltage-dependent ion channels and subsequent neurotransmitter release. alternatively, the trpm5-mediated depolarization of the basolateral membrane could modulate the function of gap junctions, and, thus change the physiological status of neighboring cells. our studies with other sensory organs strongly indicate that in addition to taste perception trpm is also involved in the processing of olfactory stimuli. accordingly, we identified trpm in two chemosensory organs, vno and olfactory epithelia. we detected trpm in solitary cells within the non-sensory epithelium and apically in the sensory epithelium of the vno. interestingly, another member of the trp gene family, the ca2+-permeable trpc is also present on the apical cell surface of the sensory epithelium of the vno. thus, it is imaginable that in conjunction with trpc trpm is required for the detection of pheromones. in this context it should be recalled that trpm cdna was isolated from the vno. furthermore, can channel activity with electrophysical properties similar to trpm or trpm was measured in freshly isolated vno neurons. preliminary findings of our group using an anti-trpm antibody illustrate that trpm is highly expressed in the sensory epithelium of the vno where it localizes to the apical region of sensory cells like trpm. these findings raise the possibility that in the vno, trpm and trpm are co-expressed and co-localized and might therefore assemble to form heteromultimeric channels whose subcellular trafficking and functional properties could differ from either channel expressed alone in other cells. in the olfactory epithelium, trpm was found in scattered cells. the morphology and distribution of trpm5-expressing cells are reminiscent of microvillar cells which are thought to be involved in sensing of odorants. by means of immunohistochemical criteria, it was recently shown that microvillar cells can be classified into at least two subtypes, one of which was denoted brush cells that were also found to be localized in the non-sensory epithelium of the vno. the physiological relevance of trpm expression in the vno and the olfactory epithelium still remains to be clarified by a meticulous analysis of odorant and pheromone sensing in trpm5-deficient mice. while this manuscript was under review, lin w. et al. reported expression data similar to ours for trpm in the main olfactory epithelium. furthermore, these authors provided evidence for an involvement of trpm in the sensing of semiochemicals, compatible with our hypothesis that trpm may take part in chemosensation. it was recently postulated that a population of specialized cells of the respiratory epithelium and gastrointestinal tract forming the so-called diffuse chemosensory system participates in the recognition and processing of diverse environmental cues. this assumption was recently substantiated by the immunolocalization of signaling proteins such as gαgust, t2r, plcβ and the ip3iii receptor in solitary intestinal epithelial cells and in cells of the respiratory system.). in accordance with these earlier findings, bezençon et al. took advantage of trpm promoter-based egfp transgenic mice and identified gfp-positive cells in the gastrointestinal tract which partially co-express a set of taste signaling proteins mentioned above. these observations are in line with our data demonstrating trpm expression in scattered epithelial cells of the respiratory system and gastrointestinal tract. the cell body of trpm5-enriched cells is bottle-shaped. the apical elongated narrow cell pole directly contacts the lumen and protrudes beyond the neighboring epithelial cells. on the basolateral cell pole, trpm5-expressing cells have a long process contacting the lamina propria. thus, the distribution and morphological characteristics of trpm5-immunopositive cells conspicuously share numerous features with brush cells. to identify the cellular origin of trpm5-immunopositive cells, we resorted to double immunofluorescence staining using antibodies directed against villin or ck proteins known to be enriched in brush cells. we observed an invariable co-localization of these marker proteins with trpm in the respiratory system and a predominant co-localization in the duodenum. brush cells were initially defined by morphological criteria, and a clear biological role of this cell population still remains elusive. hypotheses about these cells' functional role range from absorptive processes, mechanosensation, secretion or a contribution to the local immunological defense barrier. in the respiratory system and the gastrointestinal tract, brush cells may play a role in sensation of chemical compounds in the lumen. the latter notion is derived from two salient observations: firstly, brush cells display morphological features similar to trc. both cell types are characterized by an apical tuft of stiff microvilli with long rootlets into the cytoplasm lacking classical exocytotic vesicles or synapses. secondly, several signaling proteins pertinent to taste perception were identified in a subset of brush cells, including α-gustducin and no-synthase. to our knowledge, trpm is the first protein identified to be specifically expressed in brush cells of the gastrointestinal tract and the respiratory system. these results lend further support to the view that taste cells and brush cells share a common functional principle, i.e. chemosensation, and that brush cells are an important cellular correlate for chemosensation in the periphery.  the abundant and specific expression of trpm in taste receptor cells, the vno and the olfactory epithelium is consistent with the assumption that trpm has a common functional role in sensation of taste compounds, olfaction of odorants and pheromones. trpm is the first protein identified to show high and invariable enrichment in brush cells of the gastrointestinal tract and the respiratory system. our data are suggestive of brush cells as a specialized population of epithelial cells potentially involved in chemosensation and of trpm as a characteristic molecular constituent of the sensory machinery of these cells. 
a growing number of trp channels have been identified as key players in the sensation of smell, temperature, mechanical forces and taste. apart from taste buds, where we found trpm to be predominantly localized on the basolateral surface of taste receptor cells, trpm immunoreactivity was seen in other chemosensory organs – the main olfactory epithelium and the vomeronasal organ. in terms of morphological characteristics, brush cells resemble taste receptor cells, while their origin and biological role are still under intensive debate. most strikingly, we found solitary trpm5-enriched epithelial cells in all parts of the respiratory and gastrointestinal tract. based on their tissue distribution, the low cell density, morphological features and co-immunostaining with different epithelial markers, we identified these cells as brush cells. we consider trpm to be an intrinsic signaling component of mammalian chemosensory organs, and provide evidence for brush cells being an important cellular correlate in the periphery. trpm is known to be abundantly expressed in taste receptor cells where it participates in sweet, amino acid and bitter perception.
 a growing number of trp channels have been identified as key players in the sensation of smell, temperature, mechanical forces and taste. trpm is known to be abundantly expressed in taste receptor cells where it participates in sweet, amino acid and bitter perception. a role of trpm in other sensory systems, however, has not been studied so far.  here, we systematically investigated the expression of trpm in rat and mouse tissues. apart from taste buds, where we found trpm to be predominantly localized on the basolateral surface of taste receptor cells, trpm immunoreactivity was seen in other chemosensory organs – the main olfactory epithelium and the vomeronasal organ. most strikingly, we found solitary trpm5-enriched epithelial cells in all parts of the respiratory and gastrointestinal tract. based on their tissue distribution, the low cell density, morphological features and co-immunostaining with different epithelial markers, we identified these cells as brush cells. in terms of morphological characteristics, brush cells resemble taste receptor cells, while their origin and biological role are still under intensive debate.  we consider trpm to be an intrinsic signaling component of mammalian chemosensory organs, and provide evidence for brush cells being an important cellular correlate in the periphery. 