Christina A. Gurnett

Dual Function of the Voltage-Dependent Ca2+ Channel α2δ Subunit in Current Stimulation and Subunit Interaction

Abstract Voltage-dependent Ca 2+ channels are modulated by complex interactions with the α 2 δ subunit. In vitro translation was used to demonstrate a single transmembrane topology of the α 2 δ subunit in which all but the transmembrane sequence and 5 carboxy-terminal amino acids are extracellular. The glycosylated extracellular domain is required for current stimulation, as shown by coexpression of truncated α 2 δ subunits with α 1A and β 4 subunits in Xenopus oocytes and deglycosylation with peptide-N-glycosidase F. However, coexpression of the transmembrane domain-containing δ subunit reduced the stimulatory effects of full-length α 2 δ subunits and substitution of a different transmembrane domain resulted in a loss of current stimulation. These results support a model whereby the α 2 δ transmembrane domain mediates subunit interactions and the glycosylated extracellular domain enhances current amplitude.

Structural and Functional Diversity of Voltage-Activated Calcium Channels

Data gathered from the expression of cDNAs that encode the subunits of voltage-dependent Ca2+ channels have demonstrated important structural and functional similarities among these channels. Despite these convergences, there are also significant differences in the nature and functional importance of subunit-subunit and protein-Ca2+ channel interactions. There is evidence demonstrating that the functional differences between Ca2+ channel subtypes is due to several factors, including the expression of distinct alpha 1 subunit proteins, the selective association of structural subunits and modulatory proteins, and differences in posttranslational processing and cell regulation. We summarize several avenues of research that should provide significant clues about the structural features involved in the biophysical and functional diversity of voltage-dependent Ca2+ channels.

IDENTIFICATION OF THREE SUBUNITS OF THE HIGH AFFINITY OMEGA -CONOTOXIN MVIIC-SENSITIVE CA2+ CHANNEL

N-, P- and Q-type voltage-dependent Ca2+ channels control neurotransmitter release in the nervous system and are blocked by ω-conotoxin MVIIC. In this study, both a high affinity and a low affinity binding site for ω-conotoxin MVIIC were detected in rabbit brain. The low affinity binding site is shown to be present on the N-type Ca2+ channel. Using optimized conditions for specific labeling of the high affinity ω-conotoxin MVIIC receptor and a panel of subunit specific antibodies, the molecular structure of the high affinity receptor was investigated. We demonstrate for the first time that this receptor is composed of at least α1A, α2δ, and any one of the four brain β subunits. Such association of different β subunits with α1A and α2δ components may produce Ca2+ channels with distinct functional properties, such as P- and Q-type.

BETA SUBUNIT HETEROGENEITY IN N-TYPE CA2+ CHANNELS

The β subunit of the voltage-dependent Ca channel is a cytoplasmic protein that interacts directly with an α subunit, thereby modulating the biophysical properties of the channel. Herein, we demonstrate that the α subunit of the N-type Ca channel associates with several different β subunits. Polyclonal antibodies specific for three different β subunits immunoprecipitated I--conotoxin GVIA binding from solubilized rabbit brain membranes. Enrichment of the N-type Ca channels with an α subunit-specific monoclonal antibody showed the association of β, β, and β subunits. Protein sequencing of tryptic peptides of the 57-kDa component of the purified N-type Ca channel confirmed the presence of the β and β subunits. Each of the β subunits bound to the α subunit interaction domain with similar high affinity. Thus, our data demonstrate important heterogeneity in the β subunit composition of the N-type Ca channels, which may be responsible for some of the diverse kinetic properties recorded from neurons.

Extracellular Interaction of the Voltage-dependent Ca2+ Channel α2δ and α1 Subunits

The role of the extracellular domain of the voltage-dependent Ca2+ channel α2δ subunit in assembly with the α1Csubunit was investigated. Transiently transfected tsA201 cells processed the α2δ subunit properly as disulfide linkages and cleavage sites between the α2 and δ subunits were shown to be similar to native channel protein. Coimmunoprecipitation experiments demonstrated that in the absence of δ subunits, α2 subunits do not assemble with α1 subunits. Furthermore, the transmembrane and cytoplasmic sequences in δ can be exchanged with those of an unrelated protein without any effect on the association between the α2δ and α1 proteins. Extracellular domains of the α2δ subunit are also shown to be responsible for increasing the binding affinity of [3H]PN200-110 (isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-([3H]methoxycarbonyl)-pyridine-3-carboxylate) for the α1C subunit. Investigation of the corresponding interaction site on the α1 subunit revealed that although tryptic peptides containing repeat III of native α1S subunit remain in association with the α2δ subunit during wheat germ agglutinin chromatography, repeat III by itself is not sufficient for assembly with the α2δ subunit. Our results suggest that the α2δ subunit likely interacts with more than one extracellular loop of the α1 subunit.

TRANSMEMBRANE AUXILIARY SUBUNITS OF VOLTAGE-DEPENDENT ION CHANNELS

Auxiliary subunits of voltage-dependent ion channels can be subdivided into two classes. One class consists of the entirely cytoplasmic intracellular subunits, which have no transmembrane domains such as the b subunit of the voltage-dependent Ca channel and the b subunit of the voltage-dependent K channel. The other class of auxiliary subunits contains at least one transmembrane domain and an extracellular glycosylated region. This review will concentrate on the transmembrane auxiliary subunits. For mammalian systems, these proteins include the a2d and g subunits of the voltage-dependent Ca channel, the b1 and b2 subunits of the Na channel, and the b subunit of the maxi-K or large conductance Ca-activated K channel. Most traditional voltage-dependent K channels and Cl channels have not been shown to have transmembrane-containing associated proteins, although a family of membrane proteins including IsK,Cl (minK) may fit into this category. Recent genetic approaches using Drosophila and Caenorhabditis elegans have begun to identify evolutionarily related subunits as well as unique proteins that also profoundly alter ion channel properties and expression. The interest in identifying and studying these primarily extracellular proteins lies in the diverse and often unknown means by which they modify ion channel function and expression. Many are capable of altering pharmacological interactions or bind drugs directly while others are likely ligands for extracellular matrix proteins and cell-cell interactions. Some are highly negatively charged and may screen surface charges or influence ion concentration near the pore region of the ion channel. All are glycosylated to some extent suggesting a role for these proteins in plasma membrane targeting and/or subunit folding.

Intramembrane charge movements and excitation– contraction coupling expressed by two-domain fragments of the Ca2+ channel

To investigate the molecular basis of the voltage sensor that triggers excitation–contraction (EC) coupling, the four-domain pore subunit of the dihydropyridine receptor (DHPR) was cut in the cytoplasmic linker between domains II and III. cDNAs for the I-II domain (α1S 1–670) and the III-IV domain (α1S 701-1873) were expressed in dysgenic α1S-null myotubes. Coexpression of the two fragments resulted in complete recovery of DHPR intramembrane charge movement and voltage-evoked Ca2+ transients. When fragments were expressed separately, EC coupling was not recovered. However, charge movement was detected in the I-II domain expressed alone. Compared with I-II and III-IV together, the charge movement in the I-II domain accounted for about half of the total charge (Qmax = 3 ± 0.23 vs. 5.4 ± 0.76 fC/pF, respectively), and the half-activation potential for charge movement was significantly more negative (V1/2 = 0.2 ± 3.5 vs. 22 ± 3.4 mV, respectively). Thus, interactions between the four internal domains of the pore subunit in the assembled DHPR profoundly affect the voltage dependence of intramembrane charge movement. We also tested a two-domain I-II construct of the neuronal α1A Ca2+ channel. The neuronal I-II domain recovered charge movements like those of the skeletal I-II domain but could not assist the skeletal III-IV domain in the recovery of EC coupling. The results demonstrate that a functional voltage sensor capable of triggering EC coupling in skeletal myotubes can be recovered by the expression of complementary fragments of the DHPR pore subunit. Furthermore, the intrinsic voltage-sensing properties of the α1A I-II domain suggest that this hemi-Ca2+ channel could be relevant to neuronal function.

Norepinephrine release from guinea pig cardiac sympathetic nerves is insensitive to ryanodine under physiological conditions.

The activation of neurotransmitter release in nerve cells appears to be primarily dependent upon influx of extracellular Ca2+, most of which is thought to cross nerve terminal membranes through N-type Ca2+ channels. Events in skeletal and cardiac muscle, in contrast, are regulated to a greater extent by intracellular Ca2+ exchange between cytosol and intracellular organelles such as sarcoplasmic reticulum. It is not known to what extent corresponding intracellular organelles, i.e. endoplasmic reticulum (ER), contribute to cytosolic Ca2+ transients and norepinephrine (NE) release from cardiac sympathetic nerves. Heart rate and NE release were measured in isolated perfused guinea pig hearts during 1-min stimulations (5 V, 4 Hz, 2 ms) of the right stellate ganglia prior to (S1), during the administration of (S2), and after (S3) the removal of ryanodine (1 microM) from the perfusate. Ryanodine is a selective modulator of caffeine-sensitive Ca2+ stores in ER. Baseline heart rates decreased significantly in the presence of ryanodine, documenting its physiological effect on cardiac cells. However, there was no detectable effect of ryanodine on nerve-stimulated increase in heart rate or NE release. These results indicate that the ryanodine-sensitive intracellular Ca2+ stores do not play a major role in cardiac sympathetic neurotransmission.