Identification and Characterization of Novel Human Ca_v2.2 (α_1B) Calcium Channel Variants Lacking the Synaptic Protein Interaction Site

Abstract

The physical interaction between the presynaptic vesicle release complex and the large cytoplasmic region linking domains II and III of N-type (Ca(v)2.2) calcium channel alpha(1)B subunits is considered to be of fundamental importance for efficient neurotransmission. By PCR analysis of human brain cDNA libraries and IMR32 cell mRNA, we have isolated novel N-type channel variants, termed Ca(v)2.2-Delta1 and Delta2, which lack large parts of the domain II-III linker region, including the synaptic protein interaction site. They appear to be widely expressed across the human CNS as indicated by RNase protection assays. When expressed in tsA-201 cells, both novel variants formed barium-permeable channels with voltage dependences and kinetics for activation that were similar to those observed with the full-length channel. All three channel types exhibited the hallmarks of prepulse facilitation, which interestingly occurred independently of G-protein betagamma subunits. By contrast, the voltage dependence of steady-state inactivation seen with both Delta1 and Delta2 channels was shifted toward more depolarized potentials, and recovery from inactivation of Delta1 and Delta2 channels occurred more rapidly than that of the full-length channel. Moreover, the Delta1 channel was dramatically less sensitive to both omega-conotoxin MVIIA and GVIA than either the Delta2 variant or the full-length construct. Finally, the domain II-III linker region of neither variant was able to effectively bind syntaxin in vitro. These results suggest that the structure of the II-III linker region is an important determinant of N-type channel function and pharmacology. The lack of syntaxin binding hints at a unique physiological function of these channels.

Cumulative inactivation of N-type CaV2.2 calcium channels modified by alternative splicing

“…Specifically, Ca V 2.2 mRNAs containing exon 18a are abundant in peripheral ganglia, spinal cord, and in caudal regions of the brain, but are less prevalent in adult neocortex, cerebellum, and hippocampus (15,16). Alternative splicing in L II-III of Ca V 2.2 that involves exons 18a (15) or exons 19 through 21 (25) modifies channel inactivation properties without affecting the kinetics or voltage dependence of channel activation. However, it is not known whether alternative splicing in Ca V 2.2 inf luences the ability of the channel to respond to more physiologically relevant stimuli, such as a train of action potentials.…”

“…Ca V 2.2 isoforms lacking other segments of the II-III loop are more resistant to closed-state inactivation (25). The large variance associated with cumulative inactivation of Ca V 2.2e[⌬18a] currents (Fig.…”

“…The mechanism by which the inclusion of exon 18a in the II-III loop of Ca V 2.2 protects the channel from closed-state inactivation is not known, but this region clearly contains domains important for modulation of inactivation properties of the Ca V 2.2 channel (12,15,25,26). Ca V 2.2 isoforms lacking other segments of the II-III loop are more resistant to closed-state inactivation (25).…”

Interactions with PDZ Proteins Are Required for L-Type Calcium Channels to Activate cAMP Response Element-Binding Protein-Dependent Gene Expression

“…In fact, compartmentalization of calcium channels may be a common theme, because recent data have shown that the N-type calcium channel subunit ␣ 1B-1 (Ca V 2.2a) contains targeting sequences to promote its interaction with the adaptor proteins Mint/x11-like protein 1 (Mint1) and calcium/calmodulin-dependent serine protein kinase (CASK) (Maximov et al, 1999;Kaneko et al, 2002;Maximov and Bezprozvanny, 2002). The resulting interactions target N-type calcium channels to the presynaptic terminal where they play a critical role in neurotransmitter release.…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…137 P/Q-type splice variants lacking parts of the synprint region showed reduced current and a large (40 mV) rightward shift in inactivation, 138 while N-type variants with substantial deletions of the synprint motif show decreased ω-conotoxin MVIIA and GVIA sensitivity, depolarized voltage dependence of steady-state inactivation, and enhanced recovery from inactivation. 139 Another splice variant that encodes exon e18a is less sensitive to closed-state inactivation. 140 Finally, alternate splice isoforms lacking exon e19 that encodes part of the II-III linker have been reported for Ca V 2.3 that show increased sensitivity to calcium-dependent current enhancement.…”