Christopher N. Wyatt

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.

Ca 2+ Channel Currents in Type I Carotid Body Cells of Normoxic and Chronically Hypoxic Neonatal Rats

Whole-cell patch-clamp recordings were used to study voltage-gated Ca2+ channel currents in type I carotid body cells of young rats born and reared in normoxia or in a chronically hypoxic (CH) environment (10% O2). Currents activated at potentials of -40 mV and more positive, and typically peaked at 0 mV in both groups of cells. Steady-state inactivation curves were similar in the two populations. Ca2+ currents were significantly larger in CH type I cells, but this was accounted for by the increased size of CH cells: current density was similar in both cell types. Nifedipine (5 microM) always partially inhibited currents and Bay K 8644 (2-5 microM) always enhanced currents, indicating the presence of L-type channels. In a small number of cells from each group, the N-type channel blocker omega-conotoxin GVIA caused partial, irreversible inhibition, but in most cells was without discernible effect. These results indicate that type I cells possess L-type Ca2+ channels, that N-type are expressed in some cells and that non-L, non-N-type channels are also present. Furthermore, chronic hypoxia does not appear to cause specific adaptive changes in the properties of Ca2+ channels in type I cells.

Ca2+ Channel Currents in Type I Carotid Body Cells from Normoxic and Chronically Hypoxic Rats

Rats born and reared under chronically hypoxic (10% O2) conditions do not respond to acute hypoxia with an increased ventilation. Their carotid bodies undergo hyperplasia and hypertrophy and we have recently shown that K+ channels recorded in type I carotid body cells isolated from normal and chronically hypoxic (CH) rats show marked differences (Wyatt et al, 1995): normoxic type I cells express Ca2+-activated K+ (KCa) channels which are inhibited by acute hypoxia, leading to cell depolarization, opening of voltagegated Ca2+ channels (VGCCs) and the consequent influx of Ca2+ to trigger neurotransmitter release (Peers & Buckler, 1995). In type I cells from CH rats, there is far less expression of KCa channels, and, whilst the remaining K+ channels are inhibited by hypoxia, this does not lead to cell depolarization, which may explain the lack of ventilatory response to acutely inspired hypoxia in intact CH rats (Wyatt et al, 1995). An important factor in the response of normal type I cells to hypoxia is the activation of VGCCs, of which numerous sub-types exist in various tissues. It is known that L-type VGCCs are involved in hypoxic chemotransduction (Buckler & Vaughan-Jones, 1994), but very little is known about the possible presence of other VGCCs, whether they may be involved in chemotransduction, and whether they are affected by chronic hypoxia. We have therefore compared the properties of VGCCs in type I carotid body cells isolated from normoxically-reared and CH rats.