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J. Biol. Chem., Vol. 275, Issue 33, 25556-25561, August 18, 2000

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Characterization and Gene Expression of High Conductance Calcium-activated Potassium Channels Displaying Mechanosensitivity in Human Odontoblasts^*

Bruno Allard, Marie-Lise Couble^§, Henry Magloire^§¶, and Françoise Bleicher^§

From the  Laboratoire de Physiologie des Eléments Excitables, UMR CNRS 5578, Université C. Bernard Lyon I, 69622 Villeurbanne, Cedex, France and the ^§ Laboratoire du Développement des Tissus Dentaires, rue G. Paradin, EA 1892, Faculté d'Odontologie, 69372 Lyon, Cedex 08, France

Received for publication, March 20, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Odontoblasts form a layer of cells responsible for the dentin formation and possibly mediate early stages of sensory processing in teeth. Several classes of ion channels have previously been identified in the odontoblast or pulp cell membrane, and it is suspected that these channels assist in these events. This study was carried out to characterize the K_Ca channels on odontoblasts fully differentiated in vitro using the patch clamp technique and to investigate the HSLO gene expression encoding the -subunit of these channels on odontoblasts in vivo. In inside-out patches, K_Ca channels were identified on the basis of their K⁺ selectivity, conductance, voltage, and Ca²⁺ dependence. In cell-attached patches, these channels were found to be activated by application of a negative pressure as well as an osmotic shock. By reverse transcription-polymerase chain reaction, a probe complementary to K_Ca -subunit mRNA was constructed and used for in situ hybridization on human dental pulp samples. Transcripts were expressed in the odontoblast layer. The use of antibodies showed that the K_Ca channels were preferentially detected at the apical pole of the odontoblasts. These channels could be involved in mineralization processes. Their mechanosensitivity suggests that the fluid displacement within dentinal tubules could be transduced into electrical cell signals.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

During tooth development, odontoblasts originating from the neural crest are the cells responsible for the dentin formation (1). These highly polarized cells synthesize the dentin organic matrix made of collagen and noncollagenous macromolecules, which further mineralize. They play a central role in the transport and accumulation of calcium to the mineralization front (2). It is thought that the calcium pathway for dentin formation involves plasma membrane Ca²⁺ channels, because specific calcium channel blockers dramatically impair Ca²⁺ transport into dentin mineral (3). Besides these Ca²⁺ channels, Na⁺, K⁺, and recently Cl channels have also been described in human dental pulp cells cultured in vitro or odontoblasts isolated by enzyme treatment (4-6). It is suggested that these channels play a role in calcium homeostasis as well as in the cellular mechanisms underlying sensory transduction in teeth, a phenomenon that still remains unclear. Indeed, sensory axons have been identified in close contact with the odontoblast bodies or processes corresponding to the proximal end of the cells and running within the mineralized dentin tubules (for review, see Ref. 7). Thus, it is thought that a transductive mechanism for somatic sensation could exist via ion channels in odontoblasts. However, because they form a layer of cells close to the dentin, in situ electrophysiological studies of ion channels in odontoblasts as well as interaction between odontoblastic and neuronal elements proved to be impossible.

We recently developed a unique cell culture system allowing the differentiation of human dental pulp cells into odontoblasts at both the morphological and functional levels (8). We have taken advantage of this cell culture system to use the patch clamp recording techniques to examine, at the single channel level, the characteristics of ion channels in the plasma membrane of odontoblasts. We focus in this paper on high conductance Ca²⁺-activated K⁺ (K_Ca)¹ channels. Indeed, these channels have been found to be present in the plasma membrane of different cell types and implicated in the regulation of a variety of functions, such as cell firing in neurons, secretion in endocrine or exocrine cells, and myogenic tone in arterial smooth muscle (9-13). Their basic characteristic is that channel opening is induced by an increase in intracellular [Ca²⁺] as well as membrane depolarization. The gene encoding the pore-forming -subunit of this channel was first cloned in Drosophila (14) and later from various species, including human (HSLO) (15-17). We demonstrate here, in on-cell patches, that these K⁺ channels display mechanosensitivity. Furthermore, using RT-PCR, in situ hybridization, and immunohistochemistry techniques, we provide evidence for the expression and distribution of K_Ca channels at strategic locations in odontoblasts in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Odontoblast Cell Culture-- Dental pulp cells were obtained from sound human third molar germs (14-16 years old) that were extracted for orthodontic reasons. Informed consent was obtained from the patients. Explants were grown in Eagle's basal medium supplemented with ascorbic acid, antibiotics, 15% fetal calf serum, and 10 mM sodium -glycerophosphate as described previously (8). After 2-3 weeks of culture, cells exhibiting an eccentric position of the nucleus and displaying an elongated process were used either for electrophysiological recordings or harvested for isolation of total RNA. Fig. 1 shows the typical aspect of these cells under the inverted microscope.

View larger version (172K): [in this window] [in a new window]   Fig. 1.   . Micrograph of cultured human odontoblasts taken under an inverted microscope. Magnification, × 400.

Odontoblast Cell RNA Extraction and RT-PCR Analysis-- Total RNA were extracted from the cultured cells using the RNeasy kit and protocol (Qiagen, Chatsworth, CA). Purified RNA (3 µg) were reverse-transcribed using random hexamers as primers and converted into cDNA by means of the StrataScript RT-PCR kit (Stratagene, La Jolla, CA). PCR amplification was then realized from a tenth of the RT mixture in 50 µl containing 10 mM tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM dNTPs, 2 units of Taq DNA polymerase (Roche Molecular Biochemicals) and 30 pmol of each Maxi-KCa (HSLO) primer (forward primer: 5'-CAGCATTTGCCGTCAGTGTCCT-3', reverse primer: 5'-CATGCCTTTGGGTTATTTTTCC-3') corresponding to bp positions 2824-2845 and 3683-3662, respectively, of the human sequence (17). The amplification was carried out for 35 cycles (1 min at 94 °C, 1 min at 57 °C, and 1 min at 72 °C followed by 10 min at 72 °C). The PCR product was analyzed on a 1% agarose gel by electrophoresis.

In Situ Hybridization-- The material consisted of five sound nonerupted human third molars extracted for orthodontic reasons from adolescents with their informed consent. Immediately after extraction, the pulp tissue was carefully removed from the dentin walls and embedded in Tissue Tek OTC compound (EMS, Washington, PA). The specimen were then immersed in liquid nitrogen-cooled isopentane and stored frozen at 70 °C. Cryostat sections (10 µm) were collected on 3-aminopropyltriethoxysilane-coated slides, air-dried, and kept frozen (70 °C) until treatment. Culture samples were prepared as described previously in detail (8). For detection of the HSLO transcripts, in situ hybridization was performed according to Bleicher et al. (18) using a single-stranded DNA probe with a specific activity of about 2.8 × 10⁶ cpm/pmol. The images were processed using Adobe Photoshop 4.0 (Adobe Systems, San Jose, CA).

Immunohistochemistry-- Pulp slices, recovered as described above, were rinsed in PBS and incubated in PBS solution supplemented with normal goat serum (1:50, Institut Pasteur, Paris, France) and 0.2% bovine serum albumin for 30 min at room temperature. After washing, the sections were reacted with polyclonal anti-BK_Ca or anti-_1C L-type calcium channel antibodies raised in rabbit (Alomone Laboratories, Jerusalem, Israel). Subsequently, the slices were rinsed, incubated (45 min at room temperature) with Cy3 goat anti-rabbit IgG (Interchim, France), washed extensively, and sealed from the air with glycerol/PBS solution under a coverslip. Negative controls were carried out with preincubated fusion protein with antibody. Sections were examined by use of a Reichert-Jung Polyvar microscope appropriately equipped. Cultured odontoblasts were rapidly fixed with 4% paraformaldehyde, 0.05% Triton X-100 in PBS and treated as above.

Electrophysiology-- Experiments were performed on cultured odontoblasts. Single channel currents were recorded from cell-attached or inside-out membrane patches using a patch clamp amplifier (model RK 400, Bio-Logic, Claix, France). Currents flowing into the pipette were considered to be positive. Command voltage pulse generation and acquisition were done using the Biopatch software (Bio-Logic) driving an analog-to-digital, digital-to-analog converter (Lab Master DMA board, Scientific Solutions Inc., Solon, OH). Currents were analyzed using Biopatch software. Channel activity was determined from the average current (I) as NP_o = I/i in each patch, where i is the single channel current, N the number of channels in the patch, and P_o the open state probability. I was measured after filtering at 300 Hz and sampling at 1 kHz over 3-s recording periods. Single channel current amplitudes were determined using amplitude histograms.

Pipettes were pulled from borosilicate glass capillaries and had resistances of 3-4 M when filled with Tyrode solution and immersed in the K⁺-rich bathing solution. Care was taken to use gentle patches; usually, upon contact of the pipette with the cell, the release of positive pressure from the pipette was sufficient to form a gigaseal.

Solutions and Chemicals-- Pipettes were filled with Tyrode solution containing (in millimolar): 140 NaCl, 5 KCl, 2.5 CaCl₂, 1 MgCl₂, 10 Hepes, adjusted to pH 7.4 with NaOH or a K⁺-rich solution containing: 140 KCl, 2.5 CaCl₂ (or 0 Ca²⁺ plus EGTA when mentioned), 1 MgCl₂, 10 Hepes, adjusted to pH 7.4 with KOH. In cell-attached experiments, cells were bathed in the K⁺-rich solution. Saccharose was added at a concentration of 300 mM. Membrane patches were exposed to different solutions by placing them in the mouth of a perfusion tube from which flowed the rapidly exchanged solutions. Flow of the solutions did not affect channel activity.

Membrane stretch was elicited by applying negative pressure to the back end of the patch pipette through the suction port of the pipette holder. The pressure level was established by monitoring the height of water in an U-shaped tube, one end of which was at room air pressure, the other, at the desired pressure, switchable to the pipette. Step changes in pressure were produced with a valve system operated manually that switched the pipette holder between the U-tube and room air.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of K_Ca Channels in Inside-out Patches-- Upon patch excision from odontoblasts in vitro (Tyrode solution in the pipette, K⁺-rich solution containing 2.5 mM Ca²⁺ in the bath), one type of channel with a unitary current amplitude of about 6.5 pA was spontaneously active at 0 millivolt. This channel activity was totally and reversibly suppressed on perfusion of the cytoplasmic face of the patch by an internal solution containing EGTA and no added calcium (Fig. 2A) . Fig. 2B shows the conductance properties of this channel. Segments of current traces indicated that unitary current amplitudes were 2.7, 6.2, and 9 pA at 20, 0, and +20 millivolts, respectively, in the presence of 5 mM K⁺ in the pipette and 4, 4.1, and 9.7 pA at +20, 20, and 50 millivolts, respectively, in the presence of 140 mM K⁺ in the pipette (Fig. 2B). Additionally, it can be seen that channel opening increased with the depolarization amplitude; in the patch illustrated in the right panel of Fig. 2B, NP_o was 0.2, 0.65, and 0.87 at 50, 20, and +20 millivolts, respectively. In the presence of 5 mM K⁺ in the pipette, the current-voltage relationship displayed an outward rectification and indicated a conductance of 76 picosiemens at 0 millivolt (Fig. 2C). In the presence of 140 mM K⁺ the currents reversed at 0 millivolt, which is the value of the K⁺ equilibrium potential under these ionic conditions; the relationship was linear, and the best fit to the mean data indicated a conductance of 200 picosiemens (Fig. 2C). On the basis of their conductance properties, and the dependence of their activity upon intracellular calcium and voltage, these channels were identified as high conductance Ca²⁺-activated K⁺ (K_Ca) channels.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   . Identification of KCa channels in inside-out patches from odontoblasts. A, the bar indicates the period during which an internal solution devoid of Ca2+ and containing EGTA was applied. Patch potential was held at 0 millivolt. B, segment of current traces recorded at different membrane potentials (indicated next to each trace) in the presence of 5 mM K+ (left panel) and 140 mM K+ in the pipette (right panel). An internal K+-rich solution containing 2.5 mM Ca2+ was present in the bath. C, current-voltage relationships obtained in the presence of 5 mM K+ () and 140 mM K+ () in the pipette.

Mechanosensitivity of K_Ca Channels in Cell-attached Patches-- In cell-attached patches established on odontoblasts bathed in a K⁺-rich solution, brief opening of K_Ca channels (identified on the basis of their conductance: 100 picosiemens at +60 millivolts) could only be detected at highly depolarized membrane potentials, presumably because of the too low concentration of free Ca²⁺ inside the cells under these experimental conditions (Fig. 3A) . However, we observed that applying negative pressure to the membrane in cell-attached patches gave rise to a reversible increase in channel activity. In the cell-attached patch illustrated in Fig. 3B, a K⁺ solution containing EGTA and no added Ca²⁺ was present both in the pipette and in the bath. In the absence of pressure, channel activity increased with membrane depolarization. Applying suction of an amplitude of 4 kPa increased NP_o from close to zero to 0.03 at +20 millivolts, from 0.03 to 0.15 at +40 millivolts, and from 0.3 to 1.12 at +60 millivolts, respectively.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of membrane depolarization (A) and negative pressure (B) on KCa channel activity in cell-attached patches. The patch membrane potential is indicated next to each trace. In A, Tyrode solution and an internal K+-rich solution containing 2.5 mM Ca2+ were present in the pipette and in the bath, respectively, whereas in B a Ca2+-free internal K+-solution containing EGTA was present both in the pipette and in the bath. The horizontal bars indicate the period during which a negative pressure of an amplitude of 4 Pa was applied to the patch.

In cell-attached patches, at a given membrane potential, K_Ca channels were found to be activated in a pressure-dependent manner. Fig. 4A illustrates K_Ca channel activity at different negative pressures in a patch held at +20 millivolts (Tyrode solution in the pipette, K⁺-rich solution in bath). In this patch, NP_o was close to zero in control and increased to 0.4, 0.93, and 1.6 in the presence of 2, 4, and 6 kPa negative pressure amplitude, respectively. The relationship between K_Ca channel activity and negative pressure level is illustrated in Fig. 4B. The relationship was fitted with a Boltzmann equation (relative NP_o = 1/(1 + e^{(P_1/2  P)/k}), where P is the pressure applied to the pipette, P_1/2 is the amount of pressure required to induce half-maximal potentiation, and k is the steepness of the relation. The best fit to the mean data was obtained with values of 2.9 kPa for P_1/2 and of 1.1 kPa for k. Maximum activation occurred at about 6 kPa. In inside-out patches, in the presence of a low calcium concentration (0.7 µM), it was also found that negative pressure gave rise to potentiation of K_Ca channel activity (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of application of negative pressure of increasing amplitude on KCa channel activity in cell-attached patches. A, KCa channel currents recorded in the presence of different pressure levels in the pipette (indicated next to each current trace). The membrane potential was held at +20 millivolts. B, relationship between channel activity and negative pressure. Each symbol corresponds to mean ± S.E. (n = 5 patches). The curve was fitted with a Boltzmann equation (see "Results" for details).

Fig. 5 shows that an osmotic shock was also able to induce K_Ca channel opening in cell-attached patches. In control, cells were bathed in a K⁺-rich solution containing 300 mM saccharose and no channel opening was detected. Upon superfusion of the cell with a K⁺-rich solution devoid of saccharose, after a delay of about 10 s, K_Ca channels began to open and NP_o reached a maximum of 0.22. On returning to the hypertonic external solution, NP_o progressively decreased and K_Ca channels completely shut after a delay of 25 s. Similar results were obtained in four other patches.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   . Effect of an osmotic shock on KCa channel activity in a cell-attached patch. The patch potential was held at +20 millivolts. In control, an internal K+-rich solution containing 2.5 mM Ca2+ and 300 mM saccharose was present in the bath.

Detection of HSLO Transcripts by RT-PCR in Cultured Odontoblasts-- Analysis of mRNA expression using RT-PCR revealed that the -subunit was clearly expressed. The products ran at the expected size of 860 bp as shown on Fig. 6. The restriction of the  fragment gave rise to two visible fragments in close agreement with predicted sizes of 537 and 278 bp (lane 3). The 45-bp restriction fragment was too small to be detected. This confirmed that the PCR product analyzed in our studies accurately represented the HSLO sequence encoding the human K_Ca channel -subunit.

View larger version (75K): [in this window] [in a new window]   Fig. 6.   Analysis of RT-PCR products of KCa channel -subunit mRNAs from odontoblasts cultivated in vitro. Lane 1 contains the standard VIII (Roche Molecular Biochemicals). Lane 2 contains PCR product of the -subunit. The RT-PCR product migrates in the gel to a position in good agreement with its predicted size of 860 bp. Lane 3 shows restriction enzyme products of the  fragment in close agreement with predicted sizes.

In Situ Hybridization of HSLO Transcripts in Cultured Odontoblasts and Human Dental Pulp in Vivo-- As expected, HSLO transcripts were detected in cultured odontoblasts using in situ hybridization (Fig. 7a). However, we observed a relatively moderate level of transcripts in the cells despite their level of functional expression assessed by electrophysiological recordings.

View larger version (149K): [in this window] [in a new window]   Fig. 7.   a, in situ hybridization with HSLO cDNA in the cultured odontoblasts. Magnification, × 350. b, Slo protein immunostaining in the cultured odontoblasts. Positive dots are clearly detected throughout the cell bodies. Magnification, × 400.

Experiments conducted on human dental pulp tissue in vivo (Fig. 8) demonstrated that, despite a moderate level of tissue labeling, the highest density was observed in odontoblasts and pulp cells surrounding nerve fibers or arterioles.

View larger version (89K): [in this window] [in a new window]   Fig. 8.   In situ hybridization of a dental pulp section previously incubated with -32P-labeled probe complementary to HSLO mRNA. a, note the relative concentration of transcripts in the odontoblast layer (od) in contrast to pulp core (p); b, nerve fibers (nf); and c, arterioles (ar). Magnifications: a, × 300; b, × 300; c, × 280

Immunocytochemistry of K_Ca Channels in Cultured Odontoblasts and Human Dental Pulp in Vivo-- The Slo protein immunoreactivity was observed in the cultured odontoblasts with a diffuse dot staining throughout the cells (Fig. 7b). In cryostat sections of extracted pulps (Fig. 9), immunofluorescence of the BK_Ca channels showed a similar pattern of labeling in the odontoblast bodies, but the immunoreactivity was stronger at the apical pole of the cells. Nerve fibers and some blood vessel walls were also found to be positive. Anti-L-type Ca²⁺ channel antibodies gave a positive staining on odontoblasts, which was particularly intense in the apical region of the cells. Control sections showed negligible or no staining in these cells.

View larger version (115K): [in this window] [in a new window]   Fig. 9.   a, Slo immunoreactive staining in the odontoblast layer. Diffuse dots throughout the cell bodies (od), preferentially accumulated at the apical pole (arrows). Magnification, × 500. b, intense labeling of L-type calcium channels observed in the same odontoblast region. Magnification, × 500. Examples of positive immunostaining for Slo are also shown for arterioles (c) and nerve fibers (d). Magnification, × 500. e, negative control section of pulp tissue. Magnification, × 500.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we report that K_Ca channels are present in odontoblasts in culture originating from human dental pulp. Our cell culture system allowed us to unambiguously experiment on highly differentiated odontoblasts (8). We showed that odontoblast K_Ca channels are weakly active in on-cell patches even at highly depolarized membrane potentials but can be activated by applying suction in the pipette or in response to an osmotic shock applied to the cell. K_Ca channel activity was already described in human pulp cells in culture and dissociated odontoblasts from rat, but questions remain about the identity of the cells under study (4, 6). It was also demonstrated that these channels displayed mechanosensitivity. However, these data were obtained in excised patches in the presence of activating [Ca²⁺] at the cytoplasmic face, and we could not exclude that stretch sensitivity resulted from some alteration of subsarcolemmal components subsequent to patch excision. We showed that stretch activation could be revealed in the absence of calcium at the external face of the membrane (see Fig. 3B). This indicates that the increase in channel activity in response to membrane stretch was not the consequence of an influx of calcium via stretch-activated channels or stretch-induced Ca²⁺ leak. We cannot rule out a possible transduction via intracellular second messenger systems or cytosolic factors. However, the fact that stretch-induced activation was also observed in excised patches favors the hypothesis according to which the membrane stretch directly affects the odontoblast K_Ca channel protein itself or a membrane component closely related to the channel. Activation of K_Ca channels in response to membrane stretch and not mediated by an increase in Ca²⁺ entry has also been observed in osteoblast-like cells, renal cells, smooth and skeletal muscle cells, and neurons (19-23). In these preparations, channel activation was observed in the same range of pressure as in odontoblasts, i.e. half-maximal activation was achieved at pressures around 3 kPa.

In agreement with our electrophysiological data, PCR experiments and in situ hybridization performed in cultured odontoblasts demonstrated expression of the transcripts of the HSLO gene encoding the pore-forming -subunit of K_Ca channels. Additionally, the presence of the channel protein was confirmed with specific antibodies. More importantly, using in situ hybridization experiments, we showed that the HSLO gene was expressed by odontoblasts in vivo. Transcripts were also detected in nerve terminals as well as in vascular smooth muscle cells. These latter distributions are not surprising, because K_Ca channels have been shown to be involved in the regulation of smooth muscle tone and excitability of nerve terminals in central and sensory neurons (16, 24). In odontoblasts as well as in nerve terminals and vascular smooth muscle cells, a relatively weak signal was detected. Similar findings were reported by Rosenblatt et al. (25) using in situ hybridization in cochlea, nerve terminals, and vascular smooth muscle cells despite a well established functional expression of these channels in these different cell types. These results could indicate that the K_Ca channel protein undergoes a low turnover.

In vivo immunocytochemical experiments showed that the channel protein was strongly expressed at the apical pole of the cells. Interestingly, the localization correlates to the spatial distribution of L-type Ca²⁺ channels. Odontoblasts actively transport calcium to the mineralization front of the dentin matrix. This process is known to take place at the apical pole of the cell and is thought to involve voltage-dependent L-type Ca²⁺ channels (3, 26). Recently, it was reported (27) that extracellular Ca²⁺ increased intracellular free Ca²⁺ through a mechanism involving both influx of external Ca²⁺ via L-type Ca²⁺ channels as well as release of Ca²⁺ from internal stores. Colocalization of Ca²⁺ and K_Ca channels at the apical pole suggests that K_Ca channels could exert a negative control of calcium entry by hyperpolarizing odontoblasts and closing voltage-dependent Ca²⁺ channels in response to an increase in intracellular Ca²⁺.

The presence of K_Ca channels displaying mechanosensitivity in odontoblasts could also have relevance in the sensory transduction phenomenon in teeth. Mechanical stimulation of odontoblasts and fluid flow in dentinal tubules are known to elicit nociceptive responses (28, 29). Additionally, changes in osmotic gradient or probing dentin were shown to increase the discharge rate of pulp cells and evoke action potentials in their primary afferents. Afferent nerve terminals are known to coil around odontoblasts, and the close association of odontoblast processes and nerve endings (30, 31) has presupposed an interaction between these cells as the earliest step of tooth pain transmission. Stretch-activated K_Ca channels could be involved in this process. We found that K_Ca channels open in response to an osmotic shock, which was also recently found to induce an elevation of intracellular Ca²⁺ in living odontoblasts from sliced dental pulp (32). It can be postulated that, in response to mechanical stimuli, the combination of increased intracellular Ca²⁺ plus membrane stretch could cause K_Ca channel opening in odontoblasts. The resulted elevation in the extracellular [K⁺] in the restricted cleft delimited by the neuronal and odontoblast membranes may depolarize the nerve endings and lower threshold for nerve firing in the sensory tract. This could explain why K⁺-containing agents placed into deep dentinal cavities induce a brief burst of high frequency activity in the intradental nerves (33).

In conclusion, the results presented here show that in vitro differentiated human odontoblasts express mechanosensitive K_Ca channels. The HSLO transcripts coding for the -subunit constituting the pore of these channels are expressed in odontoblasts in vivo. The preferential localization of the Slo protein at the apical pole of the cells in vivo suggests a role of K_Ca channels in the mineralization process and in the mechanotransduction of fluid displacement within dentinal tubules into electrical cell signals.

	ACKNOWLEDGEMENTS

We acknowledge the staff of the "Service de stomatologie de l'hopital St Joseph" Lyon, for collecting tooth samples. We are grateful to Lee Pape for grammatical review of the manuscript.

	FOOTNOTES

^* 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.

^¶ To whom correspondence should be addressed: Tel.: 33-4-7877-8685; Fax: 33-4-7877-8757; E-mail: magloire@laennec.univ-lyon1.fr.

Published, JBC Papers in Press, June 5, 2000, DOI 10.1074/jbc.M002327200

	ABBREVIATIONS

The abbreviations used are: K_Ca, Ca²⁺-activated K⁺; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); PBS, phosphate-buffered saline; Pa, pascal(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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