Alpha-2-delta subunits of voltage-gated calcium channels

• Main Author: Hendrich, Janek K
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2008
• Subjects: Pharmacology
• ISBN: 9781303340529; 1303340526

The calcium channel alpha-2-delta (0,28) subunit is an auxiliary subunit associated with voltage-dependent calcium channels. It is implicated in the trafficking and functional expression of the calcium channel complex. This study expands the functional role of the VWA domain and the RRR motif of the 0:26 subunit, and the interaction between this subunit and the anti-epileptic drug, gabapentin The VWA domain is normally found in integrins, where it mediates binding to extracellular proteins. A mutation in the 0:28-2 subunit VWA domain (uMIDAS) did not produce the increase in current amplitude elicited by the wildtype (WT) 0:28-2 control. Co-immunoprecipitation studies using tsA-201 cells (stably expressing OC28-2 containing a mid-HA tag) co-cultured with cerebellar granule cells identified potential proteins from the cerebellar cultures that co-immunoprecipitate with the 0:28-2 protein. This suggests that protein from cerebellar cultures may bind the 0:28-2 subunit. Secondly, an RRR motif found in 0128-I subunit has been implicated as important for binding of the anticonvulsant drug gabapentin (Wang et al., 1999). The electrophysiological properties of the RRA mutant OC28 proteins were examined, and found not to enhance current amplitude to the full extent seen by WT 0:28-1 and 0:28-2 coexpression. Binding studies using both membrane and lipid raft of tsA-201 cells expressing 0:28-1 confirmed a lack of 3H-gabapentin in the R217A mutant condition. Chronic exposure of tsA-201 cells expressing Cav2.1, p4, 0:28-2 or Cav2.2, pib, 0:28-1 to gabapentin resulted in a reduction in size of the resultant currents. The inhibitory action of gabapentin was prevented by pre-incubation of cells with the system-L amino-acid transport inhibitor, suggesting gabapentin acts intracellularly after uptake via this transport mechanism. The inhibitory effects of gabapentin exposure were not replicated using co-expression of 0:28-3, or the non gabapentin-binding mutant 0128-I R217A or a28-2 R282A proteins. This indicated the inhibitory effect was mediated through gabapentin-binding calcium channel a28 subunits.