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

Abstract

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 Ca 2؉ 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.

Voltage-gated Sodium Channels Confer Excitability to Human Odontoblasts

“…Indeed in vivo, intracellular recordings have shown that odontoblasts have a resting membrane potential around Ϫ80 mV, values comparable with excitable cells (24). Previous electrophysiological studies have described the presence of ion channels in odontoblasts whose activity was modulated by mechanical stimuli (5). It is thus tempting to speculate that odontoblasts might be able to transduce and integrate diverse somatosensory signals known to elicit nociceptive responses in the pulp (drilling, dentin fluid flow, and heat and cold, for example) and initiate bursts of regenerative voltage responses.…”

“…Considering that odontoblasts are able to generate action potentials and that ␣2 and ␤2 subunits cluster at the sites of contacts between odontoblast membranes and neurites, it is tempting to propose that odontoblasts could participate in the sensory transduction process in teeth through interactions with nerve fibrils whose nature remains to be elucidated. Moreover, in vivo, nerves appear to preferentially co-localize with sodium channels at the apical pole of odontoblasts and correlates with the spatial distribution of mechanosensitive K Ca channels and L-type calcium channels (5,36). Ion channels, concentrated at this borderline between cell processes and bodies, could thus operate as molecular transducers between dentine fluid flow and the underlying layer of odontoblasts, which in turn may initiate tooth pain sensation.…”

“…However, the identity of the cultured cells under study and the expression of odontoblast key genes were not established, thus casting doubt about the cell type displaying voltage-gated sodium channel activity. To overcome these difficulties, we recently set up a unique cell culture system allowing the differentiation of human dental pulp cells into odontoblasts at the morphological, molecular, and functional levels (5,10,11). In the present study, we took advantage of this cell model to apply the patch clamp technique and determine whether a voltage-gated Na ϩ channel is functional in the odontoblast plasma membrane.…”

“…This close association suggested that odontoblasts and nerve endings may directly interact, although no synaptic structures or any junction could be detected between them (1-3). Along this line, two kinds of mechanosensitive K ϩ channels (K Ca and TREK-1) have been identified in human odontoblasts (5,6). This finding indicated that odontoblasts might be able to convert pain-evoking fluid displacement within dentinal tubules into electrical signals, strengthening their possible role as tooth sensor cells.…”

Tuning the mechanosensitivity of a BK channel by changing the linker length

“…The BK channel is one type of MS channels which is widely expressed in both excitable and non-excitable cells and plays an important role in, for example, vascular smooth muscle tone regulation, neuronal firing and endocrine cell secretion [12][13][14][15][16]. The MS properties of the BK channels expressed in skeletal muscle [17], kidney [18], smooth muscle [19], neuroepithelial cells and osteoblasts [20,21] have been characterized. We previously identified a stretch-activated BK channel (referred as SAKCaC) in cultured chick embryo ventricular myocytes [22].…”