Age-Related Homeostatic Midchannel Proteolysis of Neuronal L-type Voltage-Gated Ca2+ Channels

Abstract

SUMMARY Neural circuitry and brain activity depend critically on proper function of voltage-gated calcium channels (VGCCs), whose activity must be tightly controlled. We show that the main body of the pore-forming α1 subunit of neuronal L-type VGCCs, Cav1.2, is proteolytically cleaved, resulting in Cav1.2 fragment-channels that separate but remain on the plasma membrane. This “gmid-channel” proteolysis is regulated by channel activity, involves the Ca2+-dependent protease calpain and the ubiquitin-proteasome system, and causes attenuation and biophysical alterations of VGCC currents. Recombinant Cav1.2 fragment-channels mimicking the products of mid-channel proteolysis do not form active channels on their own, but when properly paired, produce currents with distinct biophysical properties. Mid-channel proteolysis increases dramatically with age and can be attenuated with an L-type VGCC blocker in vivo. Mid-channel proteolysis represents a novel form of homeostatic negative-feedback processing of VGCCs that could profoundly affect neuronal excitability, neurotransmission, neuroprotection, and calcium signaling in physiological and disease states.

Proteolytic processing of the L-type Ca2+ channel alpha11.2 subunit in neurons

“…We extracted forebrain slices and cortical slices from both mouse and rat for immunoprecipitation with FP1 and separation by SDS-PAGE, matching the 8% acrylamide gel conditions used in ( Michailidis et al , 2014). As expected from our earlier analysis in 7 and 9% acrylamide gels, the α 1 1.2 short form was partially separated from the long form in the 8% acrylamide gel ( Figure 4B).…”

“…We then carried out neutravidin-Sepharose pulldown and immunoblotting as described earlier ( Michailidis et al , 2014). In agreement with our findings above, CNC1 and FP1 immunoblotting of proteins in neutravidin-Sepharose pulldowns and total lysate loads separated by 8% PAGE revealed major partially separated bands at ~200–250 kDa and no evidence of a 150 kDa band ( Figure 5).…”

“…Our extensive and detailed biochemical analysis of α 1 1.2 size forms was inspired by recent work that suggested that surface α 1 1.2 is cleaved to a large degree between domains II and III ( Michailidis et al , 2014). The main evidence for the midchannel proteolysis proposed in this publication was based on immunoblotting:…”

“…More recent work has suggested that extensive proteolytic processing occurs via calpain- and ubiquitin/proteasome-mediated mechanisms that target the intracellular loop between domains II and III, yielding two prominent α 1 1.2 fragments: a 90 kDa fragment that might consist of the N-terminus and the first two integral membrane domains I and II, and a 150 kDa fragment that might consist of domains III and IV and the long C-terminus ( Michailidis et al , 2014). …”

JPH-2 interacts with Cai-handling proteins and ion channels in dyads: Contribution to premature ventricular contraction–induced cardiomyopathy

“…In PVC myocytes, NCX shifts from dyads to the lateral surface. Losing NCX in the dyad regions may be a compensatory mechanism initially (enabling higher and longer [Ca] elevations in the dyadic spaces for more sustained CICR), that can turn mal-adaptive (calpain-1 activation and cleavage of JPH-2 15 , Cav1.2 19 , and probably other dyad proteins).…”