Geração e caracterização molecular de anticorpos contra canais para sódio voltagem dependentes

Abstract

Voltage-gated sodium channels (Nav) are ion channels responsible for the initiation and propagation of action potentials in electrically excitable cells. These channels are expressed in metastatic cells of several types of cancer, and mutations associated with them are related to epileptic syndromes, skeletal myopathy, cardiac arrhythmias, neuropathies and others. Due to the important role of Na+ channels in different pathologies, the aim of this study was the production of antibodies against a conserved extracellular sequence of the Nav subfamilies. The sequence of 15 amino acids (alpha peptide) corresponding to the 11º extracellular segment located in domain IV of Nav1.1 channel, was synthesized using the FMOC method. Balb/C mice and New Zealand rabbits were immunized with the synthetic peptide conjugated to ovalbumin. Alpha peptide antibodies were tested for their ability to recognize alpha peptide in an ELISA and SPOT assay, and also for their ability to recognize the Na+ channel in HEK cells expressing Nav1.3 channels and mouse brain cortex extract. The results showed that the synthesized peptide was capable of inducing the production of site specific antibodies. Moreover, our method was effective in mapping a Na+ channel extracellular site and in identification of critical amino acids residues for the interaction with the antibody, providing new prospects for studying modulation of sodium channel and their associated pathophysiological effects