Structural and functional analysis of salivary intercalated duct cells reveals a secretory phenotype.

Currently, all salivary ducts (intercalated, striated and collecting) are assumed to function broadly in a similar manner, reclaiming ions that were secreted by the secretory acinar cells while preserving fluid volume and delivering saliva to the oral cavity. Nevertheless, there has been minimal investigation into the structural and functional differences between distinct types of salivary duct cells. Therefore, in this study, the expression profile of proteins involved in stimulus-secretion coupling, as well as the function of the intercalated duct (ID) and striated duct cells, was examined. Particular focus was placed on defining differences between distinct duct cell populations. To accomplish this, immunohistochemistry and in situ hybridization were utilized to examine the localization and expression of proteins involved in reabsorption and secretion of ions and fluid. Further, in vivo calcium imaging was employed to investigate cellular function. Based on the protein expression profile and functional data, marked differences between the IDs and striated ducts were observed. Specifically, the ID cells express proteins native to the secretory acinar cells while lacking proteins specifically expressed in the striated ducts. Further, the ID and striated duct cells display different calcium signalling characteristics, with the IDs responding to a neural stimulus in a manner similar to the acinar cells. Overall, our data suggest that the IDs have a distinct role in the secretory process, separate from the reabsorptive striated ducts. Instead, based on our evidence, the IDs express proteins found in secretory cells, generate calcium signals in a manner similar to acinar cells, and, therefore, are likely secretory cells. KEY POINTS: Current studies examining salivary intercalated duct cells are limited, with minimal documentation of the ion transport machinery and the overall role of the cells in fluid generation. Salivary intercalated duct cells are presumed to function in the same manner as other duct cells, reclaiming ions, maintaining fluid volume and delivering the final saliva to the oral cavity. Here we systematically examine the structure and function of the salivary intercalated duct cells using immunohistochemistry, in situ hybridization and by monitoring in vivo Ca2+ dynamics. Structural data revealed that the intercalated duct cells lack proteins vital for reabsorption and express proteins necessary for secretion. Ca2+ dynamics in the intercalated duct cells were consistent with those observed in secretory cells and resulted from GPCR-mediated IP3 production.

Missense mutations in inositol 1,4,5-trisphosphate receptor type 3 result in leaky Ca2+ channels and activation of store-operated Ca2+ entry.

Mutations in all subtypes of the inositol 1,4,5-trisphosphate receptor Ca2+ release channel are associated with human diseases. In this report, we investigated the functionality of three neuropathy-associated missense mutations in IP3R3 (V615M, T1424M, and R2524C). The mutants only exhibited function when highly over-expressed compared to endogenous hIP3R3. All variants resulted in elevated basal cytosolic Ca2+ levels, decreased endoplasmic reticulum Ca2+ store content, and constitutive store-operated Ca2+ entry in the absence of any stimuli, consistent with a leaky IP3R channel pore. These variants differed in channel function; when stably over-expressed the R2524C mutant was essentially dead, V615M was poorly functional, and T1424M exhibited activity greater than that of the corresponding wild-type following threshold stimulation. These results demonstrate that a common feature of these mutations is decreased IP3R3 function. In addition, these mutations exhibit a novel phenotype manifested as a constitutively open channel, which inappropriately gates SOCE in the absence of stimulation.

Simulation of Calcium Dynamics in Realistic Three-Dimensional Domains.

The cytosolic concentration of free calcium ions ([Ca2+]) is an important intracellular messenger in most cell types, and the spatial distribution of [Ca2+] is often critical. In a salivary gland acinar cell, a polarised epithelial cell, whose principal function is to transport water and thus secrete saliva, [Ca2+] controls the secretion of primary saliva, but increases in [Ca2+] are localised to the apical regions of the cell. Hence, any quantitative explanation of how [Ca2+] controls saliva secretion must take into careful account the spatial distribution of the various Ca2+ sources, Ca2+ sinks, and Ca2+-sensitive ion channels. Based on optical slices, we have previously constructed anatomically accurate three-dimensional models of seven salivary gland acinar cells, and thus shown that a model in which Ca2+ responses are confined to the apical regions of the cell is sufficient to provide a quantitative and predictive explanation of primary saliva secretion. However, reconstruction of such anatomically accurate cells is extremely time consuming and inefficient. Here, we present an alternative, mostly automated method of constructing three-dimensional cells that are approximately anatomically accurate and show that the new construction preserves the quantitative accuracy of the model.

Highly localized intracellular Ca2+ signals promote optimal salivary gland fluid secretion.

Salivary fluid secretion involves an intricate choreography of membrane transporters to result in the trans-epithelial movement of NaCl and water into the acinus lumen. Current models are largely based on experimental observations in enzymatically isolated cells where the Ca2+ signal invariably propagates globally and thus appears ideally suited to activate spatially separated Cl and K channels, present on the apical and basolateral plasma membrane, respectively. We monitored Ca2+ signals and salivary secretion in live mice expressing GCamp6F, following stimulation of the nerves innervating the submandibular gland. Consistent with in vitro studies, Ca2+ signals were initiated in the apical endoplasmic reticulum. In marked contrast to in vitro data, highly localized trains of Ca2+ transients that failed to fully propagate from the apical region were observed. Following stimuli optimum for secretion, large apical-basal gradients were elicited. A new mathematical model, incorporating these data was constructed to probe how salivary secretion can be optimally stimulated by apical Ca2+ signals.

Disease-associated mutations in inositol 1,4,5-trisphosphate receptor subunits impair channel function.

The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which form tetrameric channels, play pivotal roles in regulating the spatiotemporal patterns of intracellular calcium signals. Mutations in IP3Rs have been increasingly associated with many debilitating human diseases such as ataxia, Gillespie syndrome, and generalized anhidrosis. However, how these mutations affect IP3R function, and how the perturbation of as-sociated calcium signals contribute to the pathogenesis and severity of these diseases remains largely uncharacterized. Moreover, many of these diseases occur as the result of autosomal dominant inheritance, suggesting that WT and mutant subunits associate in heterotetrameric channels. How the in-corporation of different numbers of mutant subunits within the tetrameric channels affects its activities and results in different disease phenotypes is also unclear. In this report, we investigated representative disease-associated missense mutations to determine their effects on IP3R channel activity. Additionally, we designed concatenated IP3R constructs to create tetrameric channels with a predefined subunit composition to explore the functionality of heteromeric channels. Using calcium imaging techniques to assess IP3R channel function, we observed that all the mutations studied resulted in severely attenuated Ca2+ release when expressed as homotetramers. However, some heterotetramers retained varied degrees of function dependent on the composition of the tetramer. Our findings suggest that the effect of mutations depends on the location of the mutation in the IP3R structure, as well as on the stoichiometry of mutant subunits assembled within the tetrameric channel. These studies provide insight into the pathogenesis and penetrance of these devastating human diseases.

An inside job: Annexin 1A-Inositol 1,4,5-trisphosphate receptor interaction conveys endoplasmic reticulum luminal Ca2+ sensitivity.

Cytoplasmic Ca2+ is a pivotal regulator of IP3R activity. It is however controversial whether the [Ca2+] in the Endoplasmic Reticulum lumen also directly regulates channel function. We highlight a recent paper that demonstrates that luminal [Ca2+] potently inhibits IP3R activity. This regulation occurs indirectly by an interaction mediated through a binding partner, likely Annexin 1A.