Mounting evidence suggests that the ion pump, Na,K-ATPase, can, in the presence of ouabain, act as a signal transducer. A prominent binding motif linking the Na,K-ATPase to intracellular signaling effectors has, however, not yet been identified. Here we report that the N-terminal tail of the Na,K-ATPase catalytic α-subunit (αNT-t) binds directly to the N terminus of the inositol 1,4,5-trisphosphate receptor. Three amino acid residues, LKK, conserved in most species and most α-isoforms, are essential for the binding to occur. In wild-type cells, low concentrations of ouabain trigger low frequency calcium oscillations that activate NF-κB and protect from apoptosis. All of these effects are suppressed in cells overexpressing a peptide corresponding to αNT-t but not in cells overexpressing a peptide corresponding to αNT-tΔLKK. Thus we have identified a well conserved Na,K-ATPase motif that binds to the inositol 1,4,5-trisphosphate receptor and can trigger an anti-apoptotic calcium signal.

Coherent network activity among assemblies of interconnected cells is essential for diverse functions in the adult brain. However, cellular networks before formations of chemical synapses are poorly understood. Here, embryonic stem cell-derived neural progenitors were found to form networks exhibiting synchronous calcium ion (Ca²⁺) activity that stimulated cell proliferation. Immature neural cells established circuits that propagated electrical signals between neighboring cells, thereby activating voltage-gated Ca²⁺ channels that triggered Ca²⁺ oscillations. These network circuits were dependent on gap junctions, because blocking prevented electrotonic transmission both in vitro and in vivo. Inhibiting connexin 43 gap junctions abolished network activity, suppressed proliferation, and affected embryonic cortical layer formation. Cross-correlation analysis revealed highly correlated Ca²⁺ activities in small-world networks that followed a scale-free topology. Graph theory predicts that such network designs are effective for biological systems. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation. Significance Synchronized activity among groups of interconnected cells is essential for diverse functions in the brain. Most studies on neuronal networks have been performed in the mature brain when chemical synapses have been established. However, less is known about networking during embryonic development. We have studied neural progenitors and found that they form gap junction-mediated small-world networks, which, via electrical depolarization, drive spontaneous calcium activity to stimulate cell proliferation. Our data underscore the critical role of intricate cell signaling during embryonic development and show that complex networks of immature cells exist in the brain before birth.

Inositol 1,4,5-trisphosphate receptor type1 (IP₃R1) plays an important role in neuronal functions; however, the lateral diffusion of IP₃R1 on the endoplasmic reticulum membrane and its regulation in the living neurons remain unknown. We expressed green fluorescent protein-tagged IP₃R1 in cultured rat hippocampal neurons and observed the lateral diffusion by the fluorescence recovery after photobleaching technique. IP₃R1 showed lateral diffusion with an effective diffusion constant of ∼0.3 μm²/s. Depletion of actin filaments increased the diffusion constant of IP₃R1, suggesting that the diffusion of IP₃R1 is regulated negatively through actin filaments. We also found that protein 4.1N, which binds to IP₃R1 and contains an actin-spectrin-binding region, was responsible for this actin regulation of the IP₃R1 diffusion constant. Overexpression of dominant-negative 4.1N and blockade of 4.1N binding to IP₃R1 increased the IP₃R1 diffusion constant. The diffusion of IP₃R type 3 (IP₃R3), one of the isoforms of IP₃Rs lacking the binding ability to 4.1N, was not dependent on actin filaments but became dependent on actin filaments after the addition of a 4.1N-binding sequence. These data suggest that 4.1N serves as a linker protein between IP₃R1 and actin filaments. This actin filament-dependent regulation of IP₃R1 diffusion may be important for the spatiotemporal regulation of intracellular Ca²⁺ signaling.

Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP₃R1) using a yeast two-hybrid system. 4.1N and IP₃R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP₃R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP₃R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573–30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP₃R1 is necessary and sufficient for the localization of IP₃R1 at the basolateral membrane domain. A fragment of the IP₃R1-binding region of 4.1N blocks the localization of co-expressed IP₃R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP₃R1 translocation to the basolateral membrane domain in polarized MDCK cells.

Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca²⁺) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) are intracellular Ca²⁺ release channels that mediate Ca²⁺ release from endoplasmic reticulum (ER) Ca²⁺ stores. The three IP₃R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP₃R by the endogenous modulators IP₃, Ca²⁺, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP₃R subtype in shaping cytosolic Ca²⁺ oscillations.

Significance: The gene CACNA1C encodes for a calcium channel that has been linked to various psychiatric conditions, including schizophrenia and bipolar disorder, through hitherto unknown cellular mechanisms. Here, we report that deletion of Cacna1c in neurons of the developing brain disrupts spontaneous calcium activity and causes abnormal brain development and anxiety. Our results indicate that marginally alterations in the expression level of Cacna1c have major effects on the intrinsic spontaneous calcium activity of neural progenitors that play a crucial role in brain development. Thus, Cacna1c acts as a molecular switch that can increase susceptibility to psychiatric disease.