Glycosylation of asparagines 136 and 184 is necessary for the α 2δ subunit-mediated regulation of voltage-gated Ca 2+ channels (original) (raw)
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Journal of Biological Chemistry, 2016
Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca 2؉ channel complexes. Ca V ␣2␦1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type Ca V 1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant Ca V ␣2␦1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of Ca V ␣2␦1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the Ca V ␣2␦1-mediated increase in the peak current density and voltage-dependent gating of Ca V 1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored Ca V ␣2␦1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of Ca V ␣2␦1 as well as the Ca V ␣2␦1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of Ca V ␣2␦1, and furthermore that N-glycosylation of Ca V ␣2␦1 is essential to produce functional L-type Ca 2؉ channels.
Pflügers Archiv - European Journal of Physiology, 2013
Low-voltage-activated T-type calcium channels play important roles in neuronal physiology where they control cellular excitability and synaptic transmission. Alteration in Ttype channel expression has been linked to various pathophysiological conditions such as pain arising from diabetic neuropathy. In the present study, we looked at the role of asparagine (N)-linked glycosylation on human Ca v 3.2 T-type channel expression and function. Manipulation of N-glycans on cells expressing a recombinant Ca v 3.2 channel revealed that Nlinked glycosylation is critical for proper functional expression of the channel. Using site-directed mutagenesis to disrupt the canonical N-linked glycosylation sites of Ca v 3.2 channel, we show that glycosylation at asparagine N192 is critical for channel expression at the surface, whereas glycosylation at asparagine N1466 controls channel activity. Moreover, we demonstrate that N-linked glycosylation of Ca v 3.2 not only controls surface expression and activity of the channel but also underlies glucose-dependent potentiation of T-type Ca 2+ current. Our data suggest that N-linked glycosylation of T-type channels may play an important role in aberrant upregulation of T-type channel activity in response to glucose elevations.
Molecular Brain, 2020
Low-voltage-activated T-type calcium channels are important contributors to nervous system function. Post-translational modification of these channels has emerged as an important mechanism to control channel activity. Previous studies have documented the importance of asparagine (N)-linked glycosylation and identified several asparagine residues within the canonical consensus sequence N-X-S/T that is essential for the expression and function of Cav3.2 channels. Here, we explored the functional role of non-canonical N-glycosylation motifs in the conformation N-X-C based on site directed mutagenesis. Using a combination of electrophysiological recordings and surface biotinylation assays, we show that asparagines N345 and N1780 located in the motifs NVC and NPC, respectively, are essential for the expression of the human Cav3.2 channel in the plasma membrane. Therefore, these newly identified asparagine residues within non-canonical motifs add to those previously reported in canonical ...
Wiley Interdisciplinary Reviews: Membrane Transport and Signaling, 2013
Voltage-gated Ca 2+ (Ca V ) channels mediate Ca 2+ ions influx into cells in response to depolarization of the plasma membrane. They are responsible for initiation of excitation-contraction and excitation-secretion coupling, and the Ca 2+ that enters cells through this pathway is also important in the regulation of protein phosphorylation, gene transcription, and many other intracellular events. Initial electrophysiological studies divided Ca V channels into low-voltage-activated (LVA) and high-voltage-activated (HVA) channels. The HVA Ca V channels were further subdivided into L, N, P/Q, and R-types which are oligomeric protein complexes composed of an ion-conducting Ca V α 1 subunit and auxiliary Ca V α 2 δ, Ca V β, and Ca V γ subunits. The functional consequences of the auxiliary subunits include altered functional and pharmacological properties of the channels as well as increased current densities. The latter observation suggests an important role of the auxiliary subunits in membrane trafficking of the Ca V α 1 subunit. This includes the mechanisms by which Ca V channels are targeted to the plasma membrane and to appropriate regions within a given cell. Likewise, the auxiliary subunits seem to participate in the mechanisms that remove Ca V channels from the plasma membrane for recycling and/or degradation. Diverse studies have provided important clues to the molecular mechanisms involved in the regulation of Ca V channels by the auxiliary subunits, and the roles that these proteins could possibly play in channel targeting and membrane stabilization. /mts TABLE 1 Ca V α 1 subunits can be divided into three classes according to amino acid sequence identity, as shown in the dendrogram. The Ca V 1 and Ca V 2 classes are termed High-Voltage-Activated (HVA) channels. The Ca V 3α 1 subunits form the Low-Voltage-Activated (LVA) channels. The original names, molecular nomenclature, and type of currents are given in purple, yellow, and pink, respectively. 60 40 20 HVA LVA Amino acid identity (%) 80 100 α 1S Ca V 1.1 Ca V 1.2 Ca V 1.3 Ca V 1.4 Ca V 2.1 Ca V 2.2 Ca V 2.3 Ca V 3.1 Ca V 3.2 Ca V 3.3 α 1C L-type P/Q-type N-type 122. Harding HP, Calfon M, Urano F, Novoa I, Ron D. Transcriptional and translational control in the mammalian unfolded protein response. Annu Rev Cell Dev Biol 2002, 18:575-599.
Modulation of Cav3.2 T-type calcium channel permeability by asparagine-linked glycosylation
Channels, 2016
Low-voltage-gated T-type calcium channels are expressed throughout the nervous system where they play an essential role in shaping neuronal excitability. Defects in T-type channel expression have been linked to various neuronal disorders including neuropathic pain and epilepsy. Currently, little is known about the cellular mechanisms controlling the expression and function of T-type channels. Asparagine-linked glycosylation has recently emerged as an essential signaling pathway by which the cellular environment can control expression of T-type channels. However, the role of N-glycans in the conducting function of T-type channels remains elusive. In the present study, we used human Ca v 3.2 glycosylation-deficient channels to assess the role of N-glycosylation on the gating of the channel. Patch-clamp recordings of gating currents revealed that N-glycans attached to hCa v 3.2 channels have a minimal effect on the functioning of the channel voltage-sensor. In contrast, N-glycosylation on specific asparagine residues may have an essential role in the conducting function of the channel by enhancing the channel permeability and / or the pore opening of the channel. Our data suggest that modulation of N-linked glycosylation of hCa v 3.2 channels may play an important physiological role, and could also support the alteration of T-type currents observed in disease states.
Modulation of Ca v 3.2 T-type calcium channel permeability by asparagine-linked glycosylation
Channels, 2016
Low-voltage-gated T-type calcium channels are expressed throughout the nervous system where they play an essential role in shaping neuronal excitability. Defects in T-type channel expression have been linked to various neuronal disorders including neuropathic pain and epilepsy. Currently, little is known about the cellular mechanisms controlling the expression and function of T-type channels. Asparagine-linked glycosylation has recently emerged as an essential signaling pathway by which the cellular environment can control expression of T-type channels. However, the role of N-glycans in the conducting function of T-type channels remains elusive. In the present study, we used human Ca v 3.2 glycosylation-deficient channels to assess the role of N-glycosylation on the gating of the channel. Patch-clamp recordings of gating currents revealed that N-glycans attached to hCa v 3.2 channels have a minimal effect on the functioning of the channel voltage-sensor. In contrast, N-glycosylation on specific asparagine residues may have an essential role in the conducting function of the channel by enhancing the channel permeability and / or the pore opening of the channel. Our data suggest that modulation of N-linked glycosylation of hCa v 3.2 channels may play an important physiological role, and could also support the alteration of T-type currents observed in disease states.
The Journal of Physiology, 1998
Voltage-dependent Ca¥ channels (VDCCs) form a group of hetero-oligomeric membrane-spanning proteins (for review see Dolphin, 1995). Biophysical and pharmacological techniques have enabled native Ca¥ channel currents in many cell types to be subdivided into two major categories depending upon their kinetics and voltage-dependent properties: high voltage activated (HVA) and low voltage activated (LVA) or T-type currents (Carbone & Lux, 1984; Fedulova, Kostyuk & Veselovsky, 1985). From the initial purification studies, and the subsequent cloning of the cDNA for the constituent proteins, it became clear that calcium channels are generated by heterooligomers consisting of a pore-forming á1 subunit and two auxiliary subunits termed á2-ä and â. The cDNAs for seven á1 subunits have been cloned and functionally expressed: á1A, B, C, D, E, G and S (Perez-Reyes et al. 1998; see Perez-Reyes & Schneider, 1994, for review), and there are four genes for â subunits (Perez-Reyes & Schneider, 1994).
Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function
Trends in Neurosciences, 1998
Voltage-dependent Ca2+ channels are heteromeric complexes found in the plasma membrane of virtually all cell types and show a high level of electrophysiological and pharmacological diversity. Associated with the pore-forming alpha 1 subunit are the membrane anchored, largely extracellular alpha2-delta, the cytoplasmic beta and sometimes a transmembrane gamma subunit; these subunits dramatically influence the properties and surface expression of these channels. Effects vary depending on subunit isoforms, suggesting that functional diversity of native channels reflects heterogeneity of combinations. Interaction sites between subunits have been identified and advances have been made in our understanding of the molecular basis of functional effects of the auxiliary subunits, their capacity to be regulated by G proteins, and their interaction with related cellular systems.
Molecular Regulation of Voltage-Gated Ca 2+ Channels
Journal of Receptors and Signal Transduction, 2005
Voltage-gated Ca 2+ (Ca V ) channels are found in all excitable cells and many nonexcitable cells, in which they govern Ca 2+ influx, thereby contributing to determine a host of important physiological processes including gene transcription, muscle contraction, hormone secretion, and neurotransmitter release. The past years have seen some significant advances in our understanding of the functional, pharmacological, and molecular properties of Ca V channels. Molecular studies have revealed that several of these channels are oligomeric complexes consisting of an ion-conducting α 1 subunit and auxiliary α 2 δ, β, and γ subunits. In addition, cloning of multiple Ca V channel α 1 subunits has offered the opportunity to investigate the regulation of these proteins at the molecular level. The regulation of Ca V channels by intracellular second messengers constitutes a key mechanism for controlling Ca 2+ influx. This review summarizes recent advances that have provided important clues to the underlying molecular mechanisms involved in the regulation of Ca V channels by protein phosphorylation, G-protein activation, and interactions with Ca 2+ -binding and SNARE proteins.