Title

 

Authors

G. Cannone

Doctorate School in Biochemical and Pharmacological Sciences
Dept. of Pharmacobiology, Section of Pharmacology, Faculty of Pharmacy - University of Bari, Italy

 

Abstract

KATP channels are widely expressed in different cell types (pancreas, smooth muscle, cardiac muscle, skeletal muscle, neurons). In response to the levels of cytoplasmatic nucleotides, the activity of these channels varies correlating the energetic state of the cell to the electrical excitability. Several studies have shown that KATP channels form macromolecular complexes with proteins that contribute to the channels regulation; in cardiac myocytes and in pancreatic beta cells KATP channels are regulated by metabolic enzymes as adenylate kinase, creatine kinase, lactate dehydrogenase and pyruvate kinase (PK). However in skeletal muscle the modulation of KATP channels by PK has never been investigated. In this study we evaluated the effects of an anti-PK antibody (Ab-PK) on KATP channels from rat skeletal muscle and human Neuroblastoma cells using patch clamp techniques to verify a possible structural and functional coupling between PK and KATP channels. The possible involvment of KATP channels in the etiopathogenesis of autoimmune diseases as PANDAS has been also investigated. PANDAS, in fact, is a pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection inducing antibodies production against PK [1]. In skeletal muscle the exposure of the excised patches to all substrates of PK reaction in the absence of the Ab-PK caused a channel block by local enzymatic ATP production, the application of the antibody in the presence of all PK substrates prevented channel closure indicating a failure of the ATP production. The direct application of the Ab-PK on excised patches caused channels activation in presence of ATP and inhibition in the absence of the nucleotide. Following 1 hr incubation of the fibers with 3:1000 dilution of the antibody induced a significant enhancement of 33.5% of the KATP  currents with no changes in the fiber diameters or other cellular morphological characteristics. The long term incubation of the fibers however reduced the channel currents,  diameters and length because of channels down-regulation associated with cell death. We therefore, tested the effects of fibers incubation with anti-PK antibody negative or positive human sera (diluited 5:2000) of PANDAS patients on KATP currents. Positive serum showed an increase of IKATP 24 hours after incubation with also an increase of cell diameter and mortality rate; negative serum exerted a mild not significant increase of currents without increasing cell diameter and mortality rate in respect to controls. In neurons the Ab-PK at 3:1000 and 6:1000 dilution increased Kir/KATP currents of 38,46% and 54,81%, respectively, in respect to controls and the currents were blocked by glibenclamide 1µM. The incubation of neurons (5:2000) with the sera showed an increase of 94,56% of Kir/KATP currents 24 hours after the incubation with the positive serum and a mild not significative increase of currents with the negative serum. In conclusion, our experiments showed that Kir/KATP channels are functionally associated to PK in skeletal muscle and neuronal cells, and, since these channels are essential in regulating skeletal muscle and neuronal excitability, the lack of metabolic regulation of channels and alteration of their activity may determine skeletal muscle and neuronal consequences. We, in fact, assume that the effect of anti-PK antibody on these channels at the central and muscolar level, may influence the metabolic activity of skeletal muscle and neurons and, therefore, may be the cause of neuropsychiatric symptoms seen in patients suffering from these types of disease.
Acknowledgments: Prof. D.Tricarico, Dr. A. Mele, Prof. D.Conte Camerino,  (Dept. of Pharmacobiology, Fac. of Pharmacy, Bari), Prof. F. Vitiello and Dr. M. Buttiglione (Dept. of Biomedical Sciences and Human Oncology, Medical School, Polyclinic, Bari) are gratefully acknowledgmented.

References:
1) Dale RC (2005) - Developmental Medicine & Child Neurology, 47: 785–791