Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

National Institute Of Dental & Craniofacial Research

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Abstract

Research Advances Our key findings in this fiscal year are as follows: 1.STIM proteins, STIM1 and STIM2, play important roles in the activation of store-operated calcium entry (SOCE) through their interaction with the plasma membrane Ca2+ channel, Orai1. Unlike STIM1, STIM2 can sense small decreases in ER-Ca2+ levels, such as those induced at low stimulus intensities, and forms clusters in the ER-PM junctions. Both STIM proteins have a SAM domain as well as canonical and non-canonical EF-hand motifs in the N-terminus. The differences in their Ca2+ sensitivities are suggested to be defined by their canonical EF-hand motifs that bind Ca2+. Swapping residues in STIM1 canonical EF-hand with those of STIM2 decreases its EF-hand Ca affinity, making it STIM2-like, with increased sensitivity to low level stimuli. Conversely, interchanging STIM2 residues with those of STIM1 does not confer STIM1-like properties. Thus, the determinants of STIM2-Ca-binding affinity are yet largely unknown. The present study was designed to delineate the molecular basis for the Ca-binding affinity of STIM2 N-terminus. Towards this, we have compared the amino acid sequence of the STIM2 EF-loop with consensus sequences of this domain in other EF-hand-containing, Ca-binding proteins. We found that the canonical EF-loop of STIM2 lacks the conserved Thr and Phe at the seventh and tenth positions. Instead, it has Gly and Val. Thr at the seventh position has been suggested to promote a structure with higher Ca-binding affinity. We also found that STIM2 non-canonical EF-hand lacks several negatively charged amino acids, required for Ca2+ chelation, coordination, and geometry (amino acids at first, third, sixth, seventh and twelfth positions). Based on this, we generated a series of single, double, and triple mutants in both EF-hands that were predicted to enhance the Ca2+-binding affinity. Our preliminary results indicate that mutations in either the canonical or non-canonical EF-hand loop regions cause less basal, constitutive Ca2+ entry and ICRAC current, as compared to STIM2-WT. These mutants also display reduced clustering under resting conditions and after stimulation with low CCh, robust clustering was observed at high CCh, a response reminiscent of STIM1. Together, these results indicate that both the canonical and non-canonical EF-hands of STIM2 contribute to the Ca2+-binding affinity and basal regulatory function of STIM2. 2. Despite sharing high sequence similarity, STIM1 and STIM2 display distinct biophysical properties and differential responses to high and low stimulus intensities. STIM1 EF-hand has a high Ca2+ affinity and undergoes dramatic thermostability changes in a Ca2+-free environment. STIM2 has a relatively lower Ca2+ affinity and is relatively more thermostable in Ca2+-free conditions suggesting that the protein does not unfold as much as STIM1. Previous studies have utilized molecular dynamics (MD) simulations to investigate the conformational changes occurring in STIM1 in a Ca2+-free condition, which mimics the environment after ER-Ca2+ store depletion. These studies provided insights in understanding how STIM1 mutations affect its conformation and function. One study compared the conformational dynamics of constitutively active STIM1 mutants associated with tubular aggregate myopathy (TAM) and cancer with STIM1-WT. Although the mutations were in regions flanking the canonical EF-hand, the structure of this domain and Ca2+ affinity were altered so as to cause constitutive activation. Moreover, these mutations caused STIM1 to adopt an unfolded conformation, similar to that seen in activated STIM1 following ER-Ca2+ store depletion. The aim of our present study was to elucidate the conformational dynamics underlying STIM2 activation. Our molecular dynamics (MD) simulations studies using monomeric showed distinct N-terminal conformational changes and domain involvements that potentially contribute to STIM2 activation in response to decreases in ER-Ca2+. However, the changes in STIM2 are very subtle as compared to the large conformational unfolding that is observed for STIM1. STIM1 EF-hand mutant that resembles STIM2 displays similar STIM2-like subtle changes. These studies demonstrate that the conformational dynamics of STIM2 are distinct from those of STIM1 and are likely due to differences in their Ca2+ binding affinities and energies. Based on our MD simulations data we have also predicted potentially important regions of STIM2 N-terminus as well as residues that might contribute to their relatively stable conformation. These finding reveal as yet unidentified regions of STIM2 N-terminus that determine its regulation and response to stimuli. 3. We previously demonstrated that IR-induced salivary gland dysfunction is associated with an early transient increase in mitochondrial Ca2+ and ROSmt. Further, the mito-ROS scavenger, MitoTEMPO, treatment shortly before and on several days after IR resulted in almost complete protection of salivary gland secretion following either single (15 Gy) or fractionated (5x3 Gy) doses of irradiation. Together, these findings identified the initial increase in ROSmt, that is induced by irradiation, as a critical driver of persistent salivary gland hypofunction. We have now analyzed the mitochondrial status in irradiated cells. Our novel findings demonstrate that mitochondria undergo fission within 24 hours after irradiation and do not revert back to the original tubular morphology over the time period of the study (8 months). Importantly, mitoTEMPO treatment also protects the morphology of the mitochondria and prevents fission. Our data reveal that the mitochondria in irradiated mice salivary glands have decreased levels of ATP and display aberrant energetics. Additional analysis of the mitochondrial function will be carried out in the coming year. we hypothesize that defective autophagy might be a possible mechanism due to which the cells undergo senescence-like phenotype.