We transiently transfected the full length human pancreatic α_1D subunit, containing exon 8A in repeat I (Koschak et al. 2001), together with the α₂δ and β₃ subunits in HEK 293 cells and analysed the elementary properties of the corresponding single-channel currents.

Single α_1D channel activity could be well resolved in cell-attached patches using 100 mM BaCl₂ and 5 mM Bay K 8644. Under these conditions, α_1D channel activity was already evident at relatively low voltages (-30 mV). Step depolarizations of 200 ms to 0 mV from -80 mV revealed uniform single channel activity, characterised by brief openings (2.5 ± 0.29 ms, mean ± S.E.M., n = 25 patches) rarely alternated by long lasting ones (> 10 ms). Open probability (P_o), calculated by excluding the first and last closure of the channel and null sweeps (Carabelli et al. 2001), was 0.025 ± 0.005 (n = 4) at -30 mV and steeply increased with voltage to reach maximal activation at +20 mV (P_o 0.2 ± 0.03, n = 12), with half-maximal P_o at -14.2 mV (V_1/2). The mean open time increased from 1.2 ± 0.15 ms (-30 mV, n = 4) to 3.2 ± 0.2 ms (+10 mV, n = 12), while mean shut time decreased from 25.5 ± 2.7 ms (n = 4) to 12.8 ± 1.1 ms (n = 12). Unitary conductance was 17.7 ± 1.4 pS (n = 3Ð29). The steep voltage dependence of α_1D channel gating was also evident from the time course of averaged currents. Time to peak of mean currents was 45 ms at -30 mV and decreased to 7 ms at +10 mV (t_act < 1.5 ms). Mean amplitude progressively increased with voltage, reaching a maximal value at -10 mV. Averaged currents showed variable degrees of inactivation during pulse duration from -10 to +10 mV.

These findings are in line with whole-cell current recordings of the same α_1D subunit expressed in tsA-2001 cells (Koschak et al. 2001), characterised by low-threshold of activation (V_1/2 = -17.5 mV in 20 mM Ba), fast activation (t_act < 1 ms at 0 mV) and weak inactivation during pulses of 200 ms. Compared with the L-channel of bovine chromaffin cells, the P_o appears significantly lower (P_o,max 0.2 versus 0.56 ± 0.05, n = 14, P < 0.01, paired t test) and shifted by 20Ð25 mV toward negative voltages. The shift reduces to 3Ð4 mV when comparing the voltage dependence of P_o with the L-channel of rat insulin secreting RINm5F cells (Magnelli et al. 1996) and mouse pancreatic β-cells (Smith et al. 1993), suggesting a different distribution of α_1D subunits among different neurosecretory cells.

This work was supported by the Italian MIUR (2001.55324), the FWF (P-14820) and the EC (HPRN-CT-2000-00082).