CALHM1 and its polymorphism P86L differentially control Ca²âºhomeostasis, mitogen-activated protein kinase signaling, and cell vulnerability upon exposure to amyloid ß.

The mutated form of the Ca²âºchannel CALHM1 (Ca²âºhomeostasis modulator 1), P86L-CALHM1, has been correlated with early onset of Alzheimer's disease (AD). P86L-CALHM1 increases production of amyloid beta (Aß) upon extracellular Ca²âºremoval and its subsequent addback. The aim of this study was to investigate the effect of the overexpression of CALHM1 and P86L-CALHM, upon Aß treatment, on the following: (i) the intracellular Ca²âºsignal pathway; (ii) cell survival proteins ERK1/2 and Ca²âº/cAMP response element binding (CREB); and (iii) cell vulnerability after treatment with Aß. Using aequorins to measure the effect of nuclear Ca²âºconcentrations ([Ca²âº]n ) and cytosolic Ca²âºconcentrations ([Ca²âº]c ) on Ca²âºentry conditions, we observed that baseline [Ca²âº]n was higher in CALHM1 and P86L-CALHM1 cells than in control cells. Moreover, exposure to Aß affected [Ca²âº]c levels in HeLa cells overexpressing CALHM1 and P86L-CALHM1 compared with control cells. Treatment with Aß elicited a significant decrease in the cell survival proteins p-ERK and p-CREB, an increase in the activity of caspases 3 and 7, and more frequent cell death by inducing early apoptosis in P86L-CALHM1-overexpressing cells than in CALHM1 or control cells. These results suggest that in the presence of Aß, P86L-CALHM1 shifts the balance between neurodegeneration and neuronal survival toward the stimulation of pro-cytotoxic pathways, thus potentially contributing to its deleterious effects in AD.

Selectivity of action of pregabalin on Ca(2+) channels but not on fusion pore, exocytotic machinery, or mitochondria in chromaffin cells of the adrenal gland.

The present study was planned to investigate the action of pregabalin on voltage-dependent Ca(2+) channels (VDCCs) and novel targets (fusion pore formed between the secretory vesicle and the plasma membrane, exocytotic machinery, and mitochondria) that would further explain its inhibitory action on neurotransmitter release. Electrophysiological recordings in the perforated-patch configuration of the patch-clamp technique revealed that pregabalin inhibits by 33.4 ± 2.4 and 39 ± 4%, respectively, the Ca(2+) current charge density and exocytosis evoked by depolarizing pulses in mouse chromaffin cells. Approximately half of the inhibitory action of pregabalin was rescued by l-isoleucine, showing the involvement of α2δ-dependent and -independent mechanisms. Ca(2+) channel blockers were used to inhibit Cav1, Cav2.1, and Cav2.2 channels in mouse chromaffin cells, which were unselectively blocked by the drug. Similar values of Ca(2+) current charge blockade were obtained when pregabalin was tested in human or bovine chromaffin cells, which express very different percentages of VDCC types with respect to mouse chromaffin cells. These results demonstrate that the inhibitory action of pregabalin on VDCCs and exocytosis does not depend on α1 Ca(2+) channel subunit types. Carbon fiber amperometric recordings of digitonin-permeabilized cells showed that neither the fusion pore nor the exocytotic machinery were targeted by pregabalin. Mitochondrial Ca(2+) measurements performed with mitochondrial ratiometric pericam demonstrated that Ca(2+) uptake or release from mitochondria were not affected by the drug. The selectivity of action of pregabalin might explain its safety, good tolerability, and reduced adverse effects. In addition, the inhibition of the exocytotic process in chromaffin cells might have relevant clinical consequences.