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Importance of the Different |β Subunits in the Membrane Expression of the α1A and α2 Calcium Channel Subunits: Studies Using a Depolarization-sensitive α1A Antibody

The plasma membrane expression of the rat brain calcium channel subunits α1A, α2‐Δ and the β subunits β1b, β2a, β3b and β4 was examined by transient expression in COS‐7 cells. Neither α1A nor α2‐Δ localized to the plasma membrane, either alone or when coexpressed. However, coexpression of α1A or α2‐Δ/α1A with any of the p subunits caused α1A and α2 to be targetted to the plasma membrane. The α1A antibody is directed against an exofacial epitope at the mouth of the pore, which is not exposed unless cells are depolarized, both for native α1A channels in dorsal root ganglion neurons and for α1A expressed with a β subunit. This subsidiary result provides evidence that either channel opening or inactivation causes a conformational change at the mouth of the pore of α1A. Immunostaining for α1A was obtained in depolarized non‐permeabilized cells, indicating correct orientation in the membrane only when it was coexpressed with a subunit. In contrast, β1b and β2a were associated with the plasma membrane when expressed alone. However, this is not a prerequisite to target α1A to the membrane since β3 and β4 alone showed no differential localization, but did direct the translocation of α1A to the plasma membrane, suggesting a chaperone role for the β subunits

Functional expression of rat brain cloned alpha1E calcium channels in COS-7 cells.

Abstractu2002The properties of the rat brain α1E Ca2+ channel subunit and its modulation by accessory rat brain α2-δ and β1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether α1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca2+ channel and to examine its pharmacology in detail. α1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric α1E/α2-δ/β1b channels were largely dictated by the presence of the β1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. α1E/α2-δ/β1b channels did not appear to be regulated by Ca2+-induced inactivation. α1E was shown to exhibit a unique pharmacological profile. ω-Agatoxin IVA blocked the current in a dose-dependent manner with an IC50 of approximately 50 nM and a maximum inhibition of about 80%, whilst ω-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 μM) produced an inhibition of 51 ± 7%, whereas the DHP agonist S-(–)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the α1E Ca2+ channel subunit; we propose that rat brain α1E forms a novel Ca2+ channel with properties more similar to a subtype of LVA than HVA Ca2+ current.

Voltage‐dependent calcium channel β‐subunits in combination with α 1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by Gα o in rat frontal cortex

The dihydropyridine‐sensitive calcium channel agonist (−)‐BayK 8644 was found to produce an enhancement of the intrinsic hydrolysis of GTP by Go in rat frontal cortex membranes. An anti‐calcium channel β‐subunit antiserum abolished the (−)‐BayK 8644‐stimulated hydrolysis of GTP by Go and reduced the dihydropyridine binding capacity of the cortical membranes. A peptide which mimics the β‐subunit binding domain of the calcium channel complex, also attenuated (−)‐BayK 8644 activation of GTPase. This study suggests that the calcium channel β‐subunit is the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by Go. This may be a mechanism by which calcium channels can normally act to limit the duration of a G‐protein modulatory signal.

Properties of Cloned Rat α1A Calcium Channels Transiently Expressed in the COS‐7 Cell Line

The rat brain α1A calcium channel clone has been expressed in COS‐7 cells together with the neuronal accessory subunits p1b and α2‐δ. From reverse transcriptase polymerase chain reaction (RT‐PCR), immunocytochemistry and electrophysiology experiments, we have obtained no evidence that these cells contain any endogenous calcium channels. Transfected cells were identified by co‐expression of a cDNA for the reporter Green Fluorescent Protein. From immunocytochemical evidence, a high degree of co‐expression was obtained between Green Fluorescent Protein and individual calcium channel subunits. When all three calcium channel subunits (α1, α2‐δ and β1b) were co‐expressed, evidence was obtained that all subunits were present at the cell membrane. Voltage‐dependent calcium currents were observed between 24 and 72 h after transfection with the three calcium channel subunits. The current density for the combination α1A/α/β1ba24IPlb was 4.19 ± 0.69 pA.pF‐1 and the current produced was slowly inactivating. The time constant of inactivation of the maximum IBa was 332 ± 46 ms (n = 5). The voltage‐dependence of activation and steady‐state inactivation had voltages of half activation and inactivation of 9.5 ± 2.5 mV and ‐30.4 ± 1.5 mV respectively, and there was little overlap between the two curves. The α1A current was completely blocked by 100 μM Cd2+ and was also blocked by ω‐conotoxin MVIIC (500 nM). Dose‐inhibition curves and analysis of kon and kfor for ω‐agatoxin IVA both revealed apparent KD values of approximately 11 nM for α1A currents, with a kD of 7.8 × 104 M‐1s‐1. The results suggest that α1A expressed in these cells has some resemblance to the P type component of calcium current observed in native neurons, although it shows a somewhat greater degree of inactivation than native P current, more similar to the Q type current component. It also has an affinity for ω‐agatoxin IVA 2–5 fold lower than reported for P current, but approximately 9‐fold higher than reported for Q current.

Voltage-dependent binding and calcium channel current inhibition by an anti-α1D subunit antibody in rat dorsal root ganglion neurones and guinea-pig myocytes

1 The presence of calcium channel α1D subunit mRNA in cultured rat dorsal root ganglion (DRG) neurones and guinea‐pig cardiac myocytes was demonstrated using the reverse transcriptase‐polymerase chain reaction. 2 An antipeptide antibody targeted at a region of the voltage‐dependent calcium channel α1Dsubunit C‐terminal to the pore‐forming SS1–SS2 loop in domain IV (amino acids 1417–1434) only bound to this exofacial epitope if the DRG neurones and cardiac myocytes were depolarized with 30 mM K+. 3 Incubation of cells under depolarizing conditions for 2–4 h with the antibody resulted in a maximal inhibition of inward current density of 49% (P < 0.005) for DRGs and 30% (P < 0.05) for cardiac myocytes when compared with controls. 4 S‐(–)‐Bay K 8644 (1 μM) enhanced calcium channel currents in DRGs by 75 ± 19% (n= 5) in neurones incubated under depolarizing conditions with antibody that had been pre‐adsorbed with its immunizing peptide (100 μg ml−1). This was significantly (P < 0.05) larger than the enhancement by S‐(–)‐Bay K 8644 that was seen with cells incubated under identical conditions but with antibody alone, which was 15 ± 4% (n= 5). 5 These results demonstrate the presence of calcium channel α1D subunits in rat DRG neurones and guinea‐pig cardiac myocytes. They also show that amino acids 1417–1434 of the α1D subunit are only exposed to the extracellular face of the membrane following depolarization and that the binding of an antibody to these amino acids attenuates calcium channel current and reduces the ability of S‐(–)‐Bay K 8644 to enhance this current, indicating that it is an L‐type current that is attenuated.