Editors' ChoiceNeuroscience

Creating Separate Channel Parts

Sci. Signal.  10 Jun 2014:
Vol. 7, Issue 329, pp. ec154
DOI: 10.1126/scisignal.2005579

Voltage-dependent calcium channels (VDCCs), such as the L-type Ca2+ channels of the Cav family, control many aspects of cellular physiology. Although regulated cleavage of the C-terminal intracellular segment of Cav1.2 produces an autoinhibitory interaction, whether the many other fragments visible in Western blots of L-type VDCCs are functional or represent degradation products is unknown. Michailidis et al. identified midchannel cleavage events that occurred in the pore-forming α subunit of Cav1.2 in cortical brain slices and cultured hippocampal neurons. The authors used surface biotinylation followed by isolation and Western blotting in cortical brain slices and cultured hippocampal neurons to identify fragments that were present in the plasma membrane. Western blot analysis with antibodies directed against different epitopes of the channel indicated that the most prominent midchannel proteolysis occurred between the second and third (of the four) repeats of homologous sections that form the channel. Electrophysiological examination of neurons after washout of conditions that enhanced Cav1.2 proteolysis (increased neuronal activity or intracellular calcium) or reduced Cav1.2 proteolysis (inhibited neuronal activity) revealed that proteolysis correlated with reduced VDCC current. Image analysis of hippocampal neurons transfected with a construct expressing LGH3 [Cav1.2 tagged with green fluorescent protein (GFP) at the N terminus and hemagglutinin (HA) on an extracellular loop of the third repeat] revealed areas of the plasma membrane where the signals from the different tags on the protein (GFP and HA) were not overlapping, consistent with cleavage and separation of the two Cav1.2 fragments. Cleavage of LGH3 was reduced by exposing neurons to inhibitors of calpain, a Ca2+-activated protease, or by mutating the two ubiquitination motifs in the intracellular loops. Comparing the channel activity of LGH3 and LGH3 with a viral cleavage motif (to control cleavage by coexpression with the viral protease in Xenopus oocytes) confirmed that cleavage reduced activity. Electrophysiological analysis of Xenopus oocytes expressing individual segments of the channel that could arise from midchannel cleavage indicated that individual “half” channel segments did not form functional channels, but that cognate pairs could. Electrophysiological analysis of Xenopus oocytes coexpressing various individual segments with full-length Cav1.2 indicated that the segments differentially affected Cav1.2 channel properties. Examination of cortical slices of rats of various ages showed that midchannel proteolysis increased with age and treatment with an L-type VDCC inhibitor partially reversed the proteolysis in the oldest animals. Groth et al. predict that future studies will show that these proteolytic partial channels are not “proteinaceous detritus,” but rather may have important functions in vivo.

I. E. Michailidis, K. Abele-Henckels, W. K. Zhang, B. Lin, Y. Yu, L. S. Geyman, M. D. Ehlers, E. A. Pnevmatikakis, J. Yang, Age-related homeostatic midchannel proteolysis of neuronal L-type voltage-gated Ca2+ channels. Neuron 82, 1045–1057 (2014). [Abstract]

R. D. Groth, N. N. Tirko, R. W. Tsien, Cav1.2 calcium channels: Just cut out to be regulated? Neuron 82, 939–940 (2014). [Abstract]