TMEM16A-mediated Cl- fluxes promote contraction and set vascular tone in smooth muscle cells. In myocytes, CaMKII-mediated phosphorylation downregulates Cl- fluxes, while Ca2+-dependent phosphatases such as calcineurin (CaN) upregulate them. In HEK293 cells, we showed that increased intracellular Ca2+ promotes CaN association with FKBP12 and TMEM16A, and that the resulting complex upregulates TMEM16A activity. However, the specific binding site mediating the interaction between TMEM16A, CaN, and FKBP12 remained unknown. Here, using a stable CaN-knockdown cell line, we show that endogenous FKBP12 still co-immunoprecipitated with TMEM16A in adherent HEK293 (HEK-AD293) cells. In addition, we observed an association between TMEM16A and recombinant FKBP12 on the cytoplasmic face of plasma membranes from HEK-AD293 cells, indicating that FKBP12 can associate with TMEM16A-containing plasma membranes without an intact cytoplasmic environment. Co-localization of TMEM16A-EGFP and mRFP-FKBP12 in HEK-AD293 cells stimulated with the Ca2+ ionophore ionomycin, along with live-cell fluorescence complementation, provided fluorescence-based indirect evidence for this interaction. Finally, using GST pull-down and fluorescent complementation, we found that the TMEM16A C-terminus is necessary for FKBP12 binding. Consistent with this, C-terminally truncated TMEM16A currents were insensitive to the CaN inhibitor FK506; cyclosporin and FK506 had no effect on TMEM16A activity in CaN-depleted cells. In summary, we provide evidence that FKBP12 directly binds to the C-terminus of TMEM16A, potentially serving as a molecular anchor that recruits and localizes CaN to the channel.
Calcium-calmodulin signaling represents a central mechanism through which intracellular calcium fluctuations coordinate diverse cellular processes. Despite the identification of numerous calmodulin binding proteins, a systems level understanding of how these interactions integrate into global signaling networks and influence disease relevant pathways remains incomplete. Here, we constructed a comprehensive human calmodulin interactome by integrating experimentally validated protein-protein interactions from the IntAct and BioGRID databases, identifying 919 calmodulin associated proteins. Network analysis revealed highly connected hub proteins, including SRC and UBC, and delineated three major functional clusters enriched in translational machinery, calcium dependent kinase signaling, and actin cytoskeleton organization. Functional enrichment analyses further highlighted strong associations with phosphosignaling pathways, cytoskeletal dynamics, and hormone mediated signaling processes. Further, classification of interactome into functional categories of interest identified 102 kinases, 16 phosphatases, 24 metabolic enzymes, 29 ion channel proteins, and 56 transcription factors, underscoring the broad regulatory scope of calmodulin signaling. Integration and evaluation of publicly available calcium perturbation proteomics datasets to assess potential Ca²⁺-dependent and Ca²⁺-independent calmodulin interactions identified 43 presumed calmodulin-interacting proteins exhibiting consistent calcium-responsive regulation. Importantly, kinase substrate network mapping revealed that 46 presumed calmodulin interacting kinases phosphorylate 155 human transcription factors, suggesting formation of an extensive calmodulin linked phospho-regulatory transcriptional network. Collectively, these findings establish a systems level framework of the calmodulin signaling axis and highlight its potential relevance in transcriptional regulation, providing a resource for future mechanistic and therapeutic investigations.
Astrocytes and microglia maintain cortical homeostasis via dynamic intracellular calcium (Ca2+) signaling, but their state-dependent activity and neuronal dependencies in the auditory cortex remain poorly understood. Here, using in vivo two-photon imaging, combined with cell-type-specific viral expression of GCaMP sensors, we monitored somatic Ca2+ transients in awake and anesthetized mice. Wakefulness significantly enhanced the active cell fraction, frequency, and amplitude of somatic Ca2+ events in both glial populations compared to the anesthetized state. Local application of tetrodotoxin (TTX) completely abolished microglial Ca2+ activity, revealing an obligatory dependence on neuronal firing. In contrast, astrocytic Ca2+ events persisted at similar frequencies but exhibited significantly reduced peak amplitudes, indicating partial autonomy coupled with neuronal modulation. Together, our findings demonstrate that wakefulness coordinately boosts glial Ca2+ activity, while microglia and astrocytes rely on fundamentally distinct relationships with local neuronal networks.
TRPV3 is a heat- and ligand-gated calcium-permeable ion channel, which is mostly expressed in the skin and plays important roles in skin physiology. Targeting TRPV3 with selective ligands represents a promising therapeutic strategy for the correction of cutaneous pathological processes. For the rational drug design and prognosis of clinical outcomes, it is essential to understand the mechanisms of ligand action. Here, we study the mechanism of TRPV3 activation by cannabidiol (CBD) and inhibition by 14-deoxy-11,12-didehydroandrographolide (DDA). Pharmacological data were obtained via calcium imaging in TRPV3-expressing CHO cells and two-electrode voltage-clamp recordings in Xenopus oocytes. We show that CBD activates human TRPV3 with the EC50 of 12.8 µM, while DDA potently inhibits CBD-evoked activation with the IC50 of 7 nM. DDA acts as a modality-specific inhibitor: a competitive reversible antagonist for CBD activation, a negative allosteric modulator for 2-aminoethoxydiphenyl borate (2-APB) activation, and is neutral to heat activation. Using molecular docking and site-directed mutagenesis, we show that both CBD and DDA bind to the vanilloid site, an important regulatory pocket in the transmembrane region of TRPV3. Thus, single-residue substitutions in the vanilloid site disrupt activation by CBD (A560L) or increase its activation potency (L664S), and abrogate TRPV3 inhibition by DDA (L557A, L664S). Our results expand the pharmacological profiles of CBD and DDA and highlight their potential as drug candidates for TRPV3-targeting therapeutics.
