The role of dynamic palmitoylation in Ca ²⁺ channel inactivation

Abstract

N-and P͞Q-type Ca 2؉ channels regulate a number of critical physiological processes including synaptic transmission and hormone secretion. These Ca 2؉ channels are multisubunit proteins, consisting of a pore-forming ␣1, and accessory ␤ and ␣2␦ subunits each encoded by multiple genes and splice variants. ␤ subunits alter current amplitude and kinetics. The ␤2a subunit is associated with slowed inactivation, an effect that requires the palmitoylation of two N-terminal cysteine residues in ␤2a. In the current manuscript, we studied steady state inactivation properties of native Nand P͞Q-type Ca 2؉ channels and recombinant N-type Ca 2؉ channels. When bovine ␣1B and ␤2a and human ␣2␦ were coexpressed in tsA 201 cells, we observed significant variations in inactivation; some cells exhibited virtually no inactivation as the holding potential was altered whereas others exhibited significant inactivation. A similar variability in inactivation was observed in native channels from bovine chromaffin cells. In individual chromaffin cells, the amount of inactivation exhibited by N-type channels was correlated with the inactivation of P͞Q-type channels, suggesting a shared mechanism. Our results with recombinant channels with known ␤ subunit composition indicated that inactivation could be dynamically regulated, possibly by alterations in ␤ subunit palmitoylation. Tunicamycin, which inhibits palmitoylation, increased steady-state inactivation of Ca 2؉ channels in chromaffin cells. Cerulenin, another drug that inhibits palmitoylation, also increased inactivation. Tunicamycin produced a similar effect on recombinant N-type Ca 2؉ channels containing ␤2a but not ␤2b or ␤2a subunits mutated to be palmitoylation deficient. Our results suggest that Ca 2؉ channels containing ␤2a subunits may be regulated by dynamic palmitoylation. V oltage-dependent Ca 2ϩ channels are multimeric membrane proteins critical for a wide variety of cellular functions, including synaptic transmission, cellular growth, differentiation, and migration. Ca 2ϩ channels are classified based on their biophysical, pharmacological, and, more recently, molecular properties. Functionally, Ca 2ϩ channels have been classified as L-, N-, P͞Q-, R-and T-type; ten different ␣ 1 genes, with multiple splice variants, have been identified and called ␣ 1A -␣ 1I and ␣ 1S (1, 2). Both N-and P͞Q-type Ca 2ϩ channels have been directly linked to neurotransmitter release (3-6) and are associated with synaptic release proteins (7-11). In chromaffin cells, N-type Ca 2ϩ channels, when activated by themselves, trigger catecholamine release (12). L-and P͞Q-type channels can also initiate catecholamine release from chromaffin cells. Many chromaffin cells express an N-type current that exhibits little inactivation during long depolarizations and does not show reduced channel availability at depolarized holding potentials (13). A similar noninactivating N-type current has been described in chick ciliary ganglion (14). However, most neuronal N-type channels show robust inactivation (3), which...

Several Structural Domains Contribute to the Regulation of N-type Calcium Channel Inactivation by the β3 Subunit

“…It is unlikely that the C terminus is involved, because the Ca v 2.2⌬Xba construct was still differentially regulated by the different ␤ subunit isoforms and showed rapid inactivation in the presence of ␤ 3 . The V1 region has been previously implicated in being important for the ␤ 2a subunit-mediated slowing effects on the inactivation characteristics of Ca v 2.1, Ca v 2.3, and Ca v 1.2 calcium channel inactivation (10,(12)(13)(14). However, the ␤ 2a -mediated effects are primarily due to palmitoylation of two cysteine residues in the ␤ 2a V1 region that are present in none of the other ␤ subunits.…”

Use of analogs and inhibitors to study the functional significance of protein palmitoylation

“…The structural similarity between tunicamycin and palmitoyl CoA led to the finding that tunicamycin can also inhibit protein palmitoylation [36]. Inhibitory effects of tunicamycin on palmitoylation of GAP-43, N-type Ca++ channels, estrogen receptor α variant, and myelin proteolipid protein have been documented [30,[36][37][38].…”

CACNA2D2-mediated apoptosis in NSCLC cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity

“…Mutation of this gene has been found to lead to a phenotype characterized by epilepsy, ataxia, and alterations of calcium currents in cerebellar cells in mice, which is ultimately fatal (Barclay et al, 2001). Although the exact physiological function of CACNA2D2 in nonexcitable cells remains unknown, functional studies of CAC-NA2D2 have revealed that the activity of CACNA2D2 protein may alter the conductance properties of the pore-forming a1 unit as well as their membrane trafficking and, therefore dynamically regulates Ca 2+ current through the VACC (Gao et al, 2000;Hobom et al, 2000;Hurley et al, 2000;Klugbauer et al, 1999). The very frequent and early loss of expression of CACNA2D2 together with a subset of genes in the 3p21.3 homozygous deletion region of human chromosome 3 in human lung and breast cancers suggest a link between the CACNA2D2 and the regulation of proliferation and cell death in lung cancer pathogenesis, possibly through the regulation of the VACC-mediated Ca 2+ influx (Angeloni et al, 2000;Gao et al, 2000;Lerman and Minna, 2000).…”

“…Based on the evidence that the activity of CAC-NA2D2 dynamically regulates Ca 2+ currents in L-and T-type calcium channels Gao et al, 2000;Hobom et al, 2000;Hurley et al, 2000), we expected that overexpression of CACNA2D2 might result in an increase in the level of cytosolic Ca 2+ influx. A significant increase in the basal level of the intracellular free Ca 2+ was indeed detected in Ad-CACNA2D2-transduced H460 and A549 cells using (Berridge et al, 1998(Berridge et al, , 2000.…”