In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K(+) current (I(to)) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of I(to), even at low [Ca(2+)]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of I(to). Because channels underlying I(to) are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kvalpha-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca(2+) was used in the pipette or when intracellular [Ca(2+)] ([Ca(2+)](i)) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca(2+)-dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca(2+) increase, and the association was inhibited by KN93. These results suggest that both channels underlying I(to) are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca(2+)](i) increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca(2+)-dependent modulation by this enzyme of the associated Kv4.2 channels.

-Adrenoceptors stimulate a G ␣s protein and reduce the transient outward K ϩ current via a cAMP/PKA-mediated pathway in the rat heart.

One of the most common symptoms of diabetes is extreme hunger, but the brain mechanism underlying this hyperphagia is unknown. The endocannabinoid system has emerged as one of the main food intake regulators in the brain. However, the effects of type 1 diabetes on the endocannabinoid system are not completely known. Thus, the aim of the present work is to establish the possible alterations induced by type 1 diabetes on the brain endocannabinoid system in rats. Western blot and immunocytochemistry were used to measure CB1 and phosphorylated CB1 receptor expression in several prosencephalic regions in streptozotocin-induced type 1 diabetic rats. Serum leptin levels were measured by ELISA. CB1 receptor expression was increased in striatum and hypothalamus of diabetic animals, with no changes in other brain areas studied. CB1 receptor phosphorylation was also increased in the same brain areas. Type 1 diabetes induced significant weight loss, and serum leptin levels were severely decreased. These results reinforce the possible role of the CB1 receptor as a pharmacological target for the clinical management of appetite in diabetic patients.

Background/Aims: In diabetic ventricular myocytes, transient outward potassium current (I_to) amplitude is severely reduced because of the impaired catecholamine release that characterizes diabetic autonomic neuropathy. Sympathetic nervous system exhibits a trophic effect on I_to since incubation of myocytes with noradrenaline restores current amplitude via beta-adrenoceptor (βAR) stimulation. Here, we investigate the intracellular signalling pathway though which incubation of diabetic cardiomyocytes with the βAR agonist isoproterenol recovers I_to amplitude to normal values. Methods: Experiments were performed in ventricular myocytes isolated from streptozotocin-diabetic rats. I_to current was recorded by using the patch-clamp technique. Kv4 channel expression was determined by immunofluorescence. Protein-protein interaction was determined by coimmunoprecipitation. Results: Stimulation of βAR activates first a Gαs protein, adenylyl cyclase and Protein Kinase A. PKA-phosphorylated receptor then switches to the Gαi protein. This leads to the activation of the βAR-Kinase-1 and further receptor phosphorylation and arrestin dependent internalization. The internalized receptor-arrestin complex recruits and activates cSrc and the MAPK cascade, where Ras, c-Raf1 and finally ERK1/2 mediate the increase in Kv4.2 and Kv4.3 protein abundance in the plasma membrane. Conclusion: β₂AR stimulation activates a Gαs and Gαi protein dependent pathway where the ERK1/2 modulates the Ito current amplitude and the density of the Kv4.2 and Kv4.2 channels in the plasma membrane upon sympathetic stimulation in diabetic heart.