Figure 3. Electrophysiological characterisation of 1722-HA and 1722-HAGHA; modulation of 1722-HA with rat anti-HA antibody.
(A) Western blot analysis of tsA201 cells heterologously transfected with α2δ-1 and β3 cDNAs together with either 1722-HA or 1722-HAGHA. All subunits migrated at the expected molecular mass as indicated by an arrow. One representative experiment of at least three is shown. (B) Wild-type CaV2.1 α1, 1722-HA, or 1722-HAGHA cRNA was co-injected with auxiliary subunits into Xenopus laevis oocytes as described in section Materials and Methods. IBa was measured 1–3 days after injection (10 mM Ba2+ as charge carrier) in the indicated number of cells. Wild-type shown in black, 1722-HA in grey, and 1722-HAGHA in open symbols or dotted line. Current-voltage relationship was determined by depolarising pulses to test potentials ranging from −40 to + 30 mV from a holding potential of −80 mV. (C) Use-dependence protocol elicited 15 pulses of 100 ms in a frequency of 1 Hz from a holding potential of −60 mV to Vmax. Peak currents were normalised to peak IBa of the first pulse and are plotted against the pulse number (means ± SD). (D) Representative traces all normalised to a peak IBa of 1 μA obtained during a 3-s depolarising pulse from holding potential −80 mV to Vmax. (E) Application of 25 nM rabbit IgG as control to wild-type (black bar) and 1722-HA (grey bar), as well as 25 nM rat anti-HA antibody to 1722-HA (open bar) shown as bar graphs for the indicated number of experiments. All data are shown normalised to baseline IBa before drug application. Antisera were applied to the measured oocyte in 0.1 mg/ml BSA in bath solution. A forth experiment also showed a blocking trend but could not be quantified due to a current runup and was therefore not included in the statistical evaluation. Data are means ± SD for the indicated number of experiments. Statistical data were obtained by Student’s t test and are indicated as **p < 0.01.