Nicholas S. Berrow

Modelling of a voltage‐dependent Ca2+ channel β subunit as a basis for understanding its functional properties

Structure prediction methods have been used to establish a domain structure for the voltage‐dependent calcium channel β subunit, β1b. One domain was identified from homology searches as an SH3 domain, whilst another was shown, using threading algorithms, to be similar to yeast guanylate kinase. This domain structure suggested relatedness to the membrane‐associated guanylate kinase protein family, and that the N‐terminal domain of the β subunit might be similar to a PDZ domain. Three‐dimensional model structures have been constructed for these three domains. The extents of the domains are consistent with functional properties and mutational assays of the subunit, and provide a basis for understanding its modulatory function.

Evidence for Two Concentration-Dependent Processes for β-Subunit Effects on α1B Calcium Channels

beta-Subunits of voltage-dependent Ca(2+) channels regulate both their expression and biophysical properties. We have injected a range of concentrations of beta3-cDNA into Xenopus oocytes, with a fixed concentration of alpha1B (Ca(V)2.2) cDNA, and have quantified the corresponding linear increase of beta3 protein. The concentration dependence of a number of beta3-dependent processes has been studied. First, the dependence of the a1B maximum conductance on beta3-protein occurs with a midpoint around the endogenous concentration of beta3 (approximately 17 nM). This may represent the interaction of the beta-subunit, responsible for trafficking, with the I-II linker of the nascent channel. Second, the effect of beta3-subunits on the voltage dependence of steady-state inactivation provides evidence for two channel populations, interpreted as representing alpha1B without or with a beta3-subunit, bound with a lower affinity of 120 nM. Third, the effect of beta3 on the facilitation rate of G-protein-modulated alpha1B currents during a depolarizing prepulse to +100 mV provides evidence for the same two populations, with the rapid facilitation rate being attributed to Gbetagamma dissociation from the beta-subunit-bound alpha1B channels. The data are discussed in terms of two hypotheses, either binding of two beta-subunits to the alpha1B channel or a state-dependent alteration in affinity of the channel for the beta-subunit.

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

Abstract The 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.

Use of site-directed antibodies to probe the topography of the alpha 2 subunit of voltage-gated Ca2+ channels.

Polyclonal antibodies were raised against peptides corresponding to residues 1–15, 469–483 and 933–951 of the rabbit skeletal muscle L‐type calcium channelα2/δ primary translation product, for use as topological probes. Immunocytochemical comparison of the abilities of the antibodies to bind to theα2 and δ subunits in intact and detergent‐permeabilised rat dorsal root ganglion cells enabled the membrane orientation of these regions to be established. The resultant data indicate that the regions containing residues 1–15 and 469–483 of theα2 subunit, and residues 1–17 of the δ subunit, are exposed on the extracellular surface of the membrane, findings consistent with a model that proposesα2 to be entirely extracellular.

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.

3D Structure of the Skeletal Muscle Dihydropyridine Receptor

The dihydropyridine receptors (DHPR) are L-type voltage-gated calcium channels that regulate the flux of calcium ions across the cell membrane. Here we present the three-dimensional (3D) structure at approximately 27A resolution of purified skeletal muscle DHPR, as determined by electron microscopy and single particle analysis. Here both biochemical and 3D structural data indicate that DHPR is dimeric. DHPR dimers are composed of two arch-shaped monomers approximately 210A across and approximately 75A thick, that interact very tightly at each end of the arch. The roughly toroidal structure of the two monomers encloses a cylindrical space of approximately 80A diameter, which is then closed on each side by two dome-shaped protein densities reaching over from each monomer arch. The dome-shaped domains have a length of approximately 80-90A and a maximum height of approximately 45A. Small orifices punctuate their exterior surface. The 3D structure disclosed here may have important implications for the understanding of DHPR Ca(2+) channel function. We also propose a model for its in vivo interactions with the calcium release channel at the junctional sarcoplasmic recticulum.

The Effect of Overexpression of Auxiliary Ca2+ Channel Subunits on Native Ca2+ Channel Currents in Undifferentiated Mammalian NG108-15 Cells

1 High voltage activated (HVA) Ca2+ channels are composed of a pore‐forming α1 subunit and the accessory β and α2‐δ subunits. However, the subunit composition of low voltage activated (LVA), or T‐type, Ca2+ channels has yet to be elucidated. We have examined whether native calcium channels in NG108–15 mouse neuroblastoma × rat glioma hybrid cells, which express predominantly LVA currents when undifferentiated, are modulated by overexpression of accessory calcium channel subunits. 2 Endogenous α1A, B, C, D and E, and low levels of β and α2‐δ subunit protein were demonstrated in undifferentiated NG108–15 cells. 3 The α2‐δ, β2a or β1b accessory subunits were overexpressed by transfection of the cDNAs into these cells, and the effect examined on the endogenous Ca2+ channel currents. Heterologous expression, particularly of α2‐δ but also of β2a subunits clearly affected the profile of these currents. Both subunits induced a sustained component in the currents evoked by depolarizing voltages above −30 mV, and α2‐δ additionally caused a depolarization in the voltage dependence of current activation, suggesting that it also affected the native T‐type currents. In contrast, β1b overexpression had no effect on the endogenous Ca2+ currents, despite immunocytochemical evidence for its expression in the transfected cells. 4 These results suggest that in NG108–15 cells, overexpression of the Ca2+ channel accessory subunits α2‐δ and β2a induce a sustained component of HVA current, and α2‐δ also influences the voltage dependence of activation of the LVA current. It is possible that native T‐type α1 subunits are not associated with β subunits.

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.

Facilitation of rabbit α1B calcium channels: involvement of endogenous Gβγ subunits

1 The α1B (N‐type) calcium channel shows strong G protein modulation in the presence of G protein activators or Gβγ subunits. Using transient expression in COS‐7 cells of α1B together with the accessory subunits α2‐δ and β2a, we have examined the role of endogenous Gβγ subunits in the tonic modulation of α1B, and compared this with modulation by exogenously expressed Gβγ subunits. 2 Prepulse facilitation of control α1B/α2‐δ/β2a currents was always observed. This suggests the existence of tonic modulation of α1B subunits. To determine whether endogenous Gβγ is involved in the facilitation observed in control conditions, the βARK1 Gβγ‐binding domain (amino acids 495‐689) was overexpressed, in order to bind free Gβγ subunits. The extent of control prepulse‐induced facilitation was significantly reduced, both in terms of current amplitude and the rate of current activation. In agreement with this, GDPβS also reduced the control facilitation. 3 Co‐expression of the Gβ1γ2 subunit, together with the α1B/α2‐δ/β2a calcium channel combination, resulted in a marked degree of depolarizing prepulse‐reversible inhibition of the whole‐cell ICa or IBa. Both slowing of current activation and inhibition of the maximum current amplitude were observed, accompanied by a depolarizing shift in the mid‐point of the voltage dependence of activation. Activation of endogenous Gβγ subunits by dialysis with GTPγS produced a smaller degree of prepulse‐reversible inhibition. 4 The rate of reinhibition of α1B currents by activated G protein, following a depolarizing prepulse, was much faster with Gβ1γ2 than for the decay of facilitation in control cells. Furthermore, βARK1 (495‐689) co‐expression markedly slowed the control rate of reinhibition, suggesting that the kinetics of reinhibition depend on the concentration of free endogenous or exogenously expressed Gβγ in the cells. In contrast, the rate of loss of inhibition during a depolarizing prepulse did not vary significantly between the different conditions examined. 5 These findings indicate that, in this system, the voltage‐dependent facilitation of α1B that is observed under control conditions occurs as a result of endogenous free Gβγ binding to α1B.