In this study we have investigated properties of the membrane-anchored, but predominantly extracellular α₂δ subunit of voltage-gated Ca²⁺ channels. The α₂δ-1 and α₂δ-2 subunits enhance current amplitude, increase the rate of inactivation and hyperpolarize the steady-state inactivation of high voltage-activated (HVA) currents, although their mechanism of action is not well understood. All α₂δ subunits contain a Von Willebrand factor-A (VWF) domain within the extracellular α₂ moiety. VWF domains are present in integrins and other proteins, and contain a sequence motif representing a metal ion-dependent adhesion site (MIDAS) which confers divalent metal (usually Mg²⁺)-dependent binding to the ligand. tsA-201 and NG108-15 cells were transiently transfected using FuGENE. Green fluorescent protein was included to identify transfected cells. Where appropriate, solutions were designed to inhibit unwanted conductances. Data are displayed as % of control, mean ± S.E.M. We examined the role of the VWF domain in α₂δ-2 and in particular the importance of the MIDAS site in its functional effects. Whole-cell patch-clamp recordings from Ca_V1.2 and Ca_V2.2 channels, expressed in tsA-201 cells (H_P = -90 mV, 10 mM BaCl₂), demonstrate that deletion of the VWF domain and more specifically mutation of the three key amino acids responsible for divalent metal binding in the VWF domain (α₂δ-2 μMIDAS), completely prevented the α₂δ-2 -induced enhancement of these calcium channel currents. For example, peak Ca_V2.2 currents expressed with wild type α₂δ-2 were significantly increased to 520 ± 169% of the control in the absence of α₂δ-2 (n = 10) whereas Ca_V2.2 currents expressed with α₂δ-2 μMIDAS were unchanged at 111 ± 29% of control (n = 11). Similar results were obtained using Na⁺ as charge carrier through Ca_V2.2 channels (in the absence of any extracellular divalent cations; Ca_V2.2 (n = 25), α₂δ-2 (n = 17), α₂δ-2 μMIDAS (n = 17)). Therefore, any involvement of the MIDAS site binding of Ca²⁺ or Mg ²⁺, in the function of α₂δ-2, in relation to Ca_Vα1 subunit expression or function, is likely to be during channel assembly or trafficking to the membrane, rather than once the channel has reached the cell surface. Furthermore, although the wild type α₂δ-2 enhanced endogenous HVA calcium currents (n = 13) in differentiated NG108-15 cells (BaCl₂ 20 mM, H_P = -40 mV) to 209 ± 39% of control (n = 13), the α₂δ-2 μMIDAS construct had no significant effect, the current being 92 ± 16% of control (n = 12). Both immunocytochemical and cell surface biotinylation experiments show that these mutations in α₂δ-2 do not prevent their expression at the cell surface. Our future experiments will aim to determine how the mutations in the MIDAS site affect calcium channel function.