Proteolytic processing of the L-type Ca ²⁺ channel alpha ₁1.2 subunit in neurons

Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α ₁1.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α ₁1.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014). Methods: To better determine the apparent molecular masses (M _R)of the α ₁1.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α ₁1.2 expressed in HEK293 cells was conducted using six different region-specific antibodies against α ₁1.2. Results: The full length form of α ₁1.2 migrated, as expected, with an apparent M _R of ~250 kDa. A shorter form of comparable prevalence with an apparent M _R of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α ₁1.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α ₁1.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014) is at best minimal in brain tissue.