Structural and Functional Studies of the a2d voltage-gated Calcium Channel Subunit
Final Report Abstract
The extracellular region of the transmembrane voltage-gated calcium channel Cavα2δ subunit is the target for drugs as gabapentin (Neurotonin) and pregabalin (Lyrica) that are administered in treatment of neuropathic pain, epilepsy, fibromyalgia and other neuronal system disorders. Despite this direct connection, nothing is known about the Cavα2δ molecular structure at high resolution. The work of this project provides prerequisites towards the elucidation of the Cavα2δ three-dimensional structure. Several expression systems were assessed to find the most suitable for obtaining large quantities of purified, homogenous and functional protein required for crystallization. Extracellular Cavα2δ constructs expressed in a mammalian system lacking a specific enzyme for the processing of glycosylation indicated homogeneity in their glycosylation pattern. Secreted Cavα2δ protein was purified by basic one-step purification and showed already a high degree of purity. This material was applied for biochemical characterization such as glycosylation properties and functional studies. As a functional assay, a radio-ligand binding assay using [3H]-Gabapentin was set up and showed affinities of the recombinant Cavα2δ comparable to those reported in the literature. The results of the performed studies suggest that the purified material is native-like and hence suitable for crystallization screening.
