Calmodulin binds and modulates K+-dependent Na+/Ca2+-exchanger isoform 4, NCKX4.

The family of K+-dependent Na+/Ca2+-exchangers, NCKX, are important mediators of cellular Ca2+ efflux, particularly in neurons associated with sensory transduction. The NCKX family comprises five proteins, NCKX1–5, each the product of a different SLC24 gene. NCKX4 (SLC24A4) has been found to have a critical role in termination and adaptation of visual and olfactory signals, melanocortin-dependent satiety signaling, and the maturation of dental enamel. To explore mechanisms that might influence the temporal control of NCKX4 activity, a yeast two-hybrid system was used to search for protein interaction partners. We identified calmodulin as a partner for NCKX4, and confirmed the interaction using glutathione-S-transferase-fusion-pulldown. Calmodulin binding to NCKX4 was demonstrated in extracts from mouse brain and in transfected HEK293 cells. Calmodulin bound in a Ca2+-dependent manner to a motif present in the central cytosolic loop of NCKX4, and was abolished by the double mutant I328D/F334D. When co-transfected in HEK293 cells, calmodulin bound to NCKX4 under basal conditions and induced a ~2.5-fold increase in NCKX4 abundance, but did not influence either cellular location or basal activity. When purinergic stimulation of NCKX4 was examined in these cells, co-expression of wild type calmodulin, but not a Ca2+ binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca2+ binding to calmodulin pre-positioned on NCKX4 induces a slow conformational rearrangement that interferes with purinergic stimulation of the exchanger, possibly by obscuring T331, a previously identified potential protein kinase C site.

Abstract P006: Purinergic Receptor Activation Protects Glomerular Microvasculature From Increased Mechanical Stress In Angiotensin Ii-induced Hypertension: A Modeling Study

Angiotensin II (Ang II)-induced hypertension increases afferent and efferent arteriole resistances via the actions of Ang II on the AT1 receptor. In addition to the increased interstitial levels of Ang II, the increased arterial pressure increases interstitial ATP concentrations which act on the purinergic receptors P2X1 and P2X7, to constrict the AA, preventing increases in plasma flow and single nephron GFR (SNGFR). Blockade of the P2 receptors also mitigates the effects of Ang II, thus increasing blood flow and SNGFR, but the resulting increases in mechanical stresses (shear stress and circumferential hoop stress) on the glomerular microvasculature have not been quantified. A mathematical microvascular hemodynamic glomerular model was developed to simulate blood flow and plasma filtration at each of 320 capillary segments in an anatomically-accurate rat glomerular capillary network topology. Afferent and efferent arteriole resistances and network hydraulic conductivity were adjusted to match glomerular hemodynamic data for control, Ang II-induced hypertension and P2X1-blocked conditions (Franco, Martha, et al. Amer. J. Physiology-Renal 313.1 (2017): F9-F19). Ang II infusion increased both afferent and efferent resistances, reducing blood flow while slightly raising glomerular pressure. Blockade of the purinergic receptors reduced both afferent and efferent resistances, maintaining glomerular pressure at hypertensive levels but increasing blood flow significantly, increasing shear stress from 24.9 dynes/cm 2 in hypertensive conditions to 71.3 dynes/cm 2 after purinergic blockade. Because glomerular pressure was maintained, hoop stress barely changed from 69.5 kPa in hypertensive conditions to 70.9 kPa after purinergic blockade. Purinergic blockade also increased hydraulic conductivity and filtering surface area, increasing SNGFR. In hypertension, purinergic stimulation does not prevent the transmission of increased arterial pressure to the glomerular capillaries to reduce capillary hoop stress. However, activation of the purinergic system protects the glomerular microvasculature from increases in shear stress caused by a marked increase in blood flow that would occur in the absence of purinergic stimulation.

Purinergic Calcium Signals in Tumor-Derived Endothelium

Tumor microenvironment is particularly enriched with extracellular ATP (eATP), but conflicting evidence has been provided on its functional effects on tumor growth and vascular remodeling. We have previously shown that high eATP concentrations exert a strong anti-migratory, antiangiogenic and normalizing activity on human tumor-derived endothelial cells (TECs). Since both metabotropic and ionotropic purinergic receptors trigger cytosolic calcium increase ([Ca2+]c), the present work investigated the properties of [Ca2+]c events elicited by high eATP in TECs and their role in anti-migratory activity. In particular, the quantitative and kinetic properties of purinergic-induced Ca2+ release from intracellular stores and Ca2+ entry from extracellular medium were investigated. The main conclusions are: (1) stimulation of TECs with high eATP triggers [Ca2+]c signals which include Ca2+ mobilization from intracellular stores (mainly ER) and Ca2+ entry through the plasma membrane; (2) the long-lasting Ca2+ influx phase requires both store-operated Ca2+ entry (SOCE) and non-SOCE components; (3) SOCE is not significantly involved in the antimigratory effect of high ATP stimulation; (4) ER is the main source for intracellular Ca2+ release by eATP: it is required for the constitutive migratory potential of TECs but is not the only determinant for the inhibitory effect of high eATP; (5) a complex interplay occurs among ER, mitochondria and lysosomes upon purinergic stimulation; (6) high eUTP is unable to inhibit TEC migration and evokes [Ca2+]c signals very similar to those described for eATP. The potential role played by store-independent Ca2+ entry and Ca2+-independent events in the regulation of TEC migration by high purinergic stimula deserves future investigation.

Purinergic stimulation of K+-dependent Na+/Ca2+ exchanger isoform 4 requires dual activation by PKC and CaMKII

Activity of the K+-dependent Na+/Ca2+-exchanger, NCKX4, is stimulated by purinergic signals that depend on dual activation of two protein kinase pathways. This regulation provides a novel mechanism to shape Ca2+ signaling and thus to have important impact on neuronal processes.