Inhibitory effect of prilocaine on the ATP-dependent Ca-ATPase

Objectives: Sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) is the most efficient system of muscle to maintain [Ca²⁺] under physiological conditions. It transports 2Ca²⁺/1mol ATP into the SR lumen, inducing muscle relaxation. Its Inhibition by prilocaine (P) could account for physiophatological conditions. The aim of this work was to describe the effect of P on SERCA activity and to identify the sites of its amino acid sequence involved in such effect.
Methods: SR membranes from fast-twitch skeletal muscle with calcium accumulation ability were obtained as sealed vesicles by differential centrifugation. The ATPase activity was measured by a colorimetric method. The P half-maximal inhibitory concentration (K_i) was determined (mean±SD) and evaluated by ANOVA (p<0.05). RNA was extracted and mRNA separated by chromatography, from which cDNA was obtained by RT-PCR in the presence of 30 mM prilocaine and amplified by RACE-PCR. Deoxinucleotides sequencing method was used for cDNA sequencing. The DNA sequence was analyzed using MacDNASIS and its homology compared to GenBank® using BLAST® algorithm.
Results: In the presence and absent of calcimycin (C), P inhibited ATPase activity in the studied range (3-90 mM). Activity values were significantly higher in the presence of C than in its absence even at high [P]. K_i values (mM P) as a function of [ATP] decreased up to a constant value with C (75.0±2.6; 22.1±2.3), and without C (28.3±2.8; 14.0±0.3) for 0.5 and 4 mM ATP, showing a statistical significant difference. The isolated SERCA1a revealed a nucleotide sequence of 3000 bp and 1000 amino acids (100-110 kDa). P altered the amino acid sequence at the ATP binding site (aa 697, 710-712) and the phosphorylation site (aa 348, 352-353).
Conclusions: The inhibitory effect of P on SERCA activity in fast-twitch muscles is modulated by ATP and relates to direct alteration of specific amino acids of functional sites of the enzyme. Grant UBACyT20020110100082.