Yasuda T, Lewis RJ, Adams DJ.

	Figure 1. . The β3 subunit markedly suppressed the current amplitude of N-type (Cav2.2; A and B) and R-type (Cav2.3; C and D), but not L-type (Cav1.2; E and F) calcium channels at physiological HPs in Xenopus oocytes. For N- and R-type channels, cells were injected with cRNAs (cDNA for Cav2.3α1) encoding either Cav2.2α1 (2.5 ng/cell) or Cav2.3α1 (4.5 ng/cell) and α2δ1 (2.5 ng/cell) in combination with β3 (1.25, 2.5, or 12.5 ng/cell). Cells expressed L-type channels were injected with cRNA encoding Cav1.2α1 (5 ng/cell) and α2δ1 (12.5 ng/cell) in combination with β3 (5 or 25 ng /cell). Currents were evoked by a brief depolarization to 0 mV from HPs of −60 and −80 mV. (A, C, and E) Superimposed current traces of whole cell Ba2+ currents through the calcium channels. Bars, 0.5 μA and 50 ms. Residual capacitance transients after leak subtraction have been erased. (B, D, and F) Summary of the effects of the β3 subunit on the peak currents of N- (n = 9), R- (n = 7), and L-type (n = 4) calcium channels. Data are indicated as mean ± SE. Asterisks denote significant difference between any two groups in each set of experiments (**P < 0.01; one-way ANOVA with Bonferroni's multiple comparison test for B and D and unpaired t-test for F).
	Figure 2. . The β3 subunit caused a biphasic effect on N-type calcium channels at a physiological HP. Oocytes were injected with cRNAs encoding Cav2.2α1 (2.5 ng/cell) and α2δ1 (2.5 ng/cell) in combination with various concentrations of β3 (0, 0.5, 1.25, 2.5, 5, or 12.5 ng/cell). (A) Examples of whole cell Ba2+ currents at HPs of −80 mV (left) and −120 mV (right). (B) Corresponding I-V relationships for peak Ba2+ currents at HPs of −80 (open circle) and −120 mV (closed circle). Activation midpoints (V1/2, act at the HPs of −80 and −120 mV) calculated from the Boltzmann fitting are: β3 0 ng, −2.7 and −3.0 mV; 0.5 ng, −3.4 and −5.8 mV; 1.25 ng, −4.1 and −8.5 mV; 2.5 ng, −5.9 and −12.4 mV; 5.0 ng, −6.8 and −13.3 mV; 12.5 ng, −10.7 and −14.7 mV, respectively. (C) Effects of the β3 subunit on the maximum conductance (Gmax) at the HPs of −80 (open column) and −120 mV (closed column) and the Gmax ratio (hatched column). Data are indicated as mean ± SE (n = 11–12 from two frogs). Asterisks denote significant difference versus the control group without β3 (***P < 0.001; one-way ANOVA with Dunnett's multiple comparison test).
	Figure 3. . The β3-induced hyperpolarizing shift of the steady-state inactivation curves of N-type calcium channels is attributed to change in the proportion of two components of the curves. Oocytes were injected with cRNAs as shown in Fig. 2. (A) Steady-state inactivation for HPs of 3-min duration. Data are indicated as mean ± SE (n = 7–15 from 3–7 frogs for each group). Note that HPs of 3 min did not induce maximal inactivation and therefore reflect “pseudo-steady-state” inactivation (compare Fig. 8). (B) Effects of the β3 subunit on inactivation midpoints (V1/2, inact) of LVI (open circle) and HVI (open triangle) and the proportion of LVI (%LVI; closed column). Plotted data were derived from the curve fitting in panel A and shown as mean ± SE. (C) Difference in concentration dependency of the β3-induced enhancement of Gmax and increase in the %LVI. Plotted data were derived from B and Fig. 2 C for %LVI and Gmax at a HP of −120 mV, respectively. Data were normalized by dividing each value by the maximum value and are expressed as mean ± SE. (D) No or little overlap of activation and inactivation curves seen in the presence or absence of β3. Plotted data were derived from panel A and Fig. 2 B.
	Figure 4. . The oocyte β3 (β3xo) subunit injected exogenously with N-type calcium channels modified channel properties similar to that observed for the rat β3 subunit. Oocytes were injected with cRNAs encoding Cav2.2α1 (2.5 ng/cell) and α2δ (2.5 ng/cell) subunits in combination with various concentrations of β3xo subunit (0, 0.1, 0.5, or 2.5 ng/cell). (A) I-V relationships for peak Ba2+ currents at HPs of −80 (open circle) and −120 mV (closed circle). (B) Effects of the β3xo subunit on the maximum conductance (Gmax) at HPs of −80 mV (open column) and −120 mV (closed column) and Gmax ratio (hatched column). (C) The β3xo caused a leftward shift of the steady-state inactivation curve. Inactivation midpoints (V1/2, inact) were as follows: β3xo 0 ng (LVI and HVI), −72.6 and −45.5 mV; 0.5 ng, −74.7 and −42.1 mV; 2.5 ng, −75.4 and −40.0 mV. Proportions of LVI were as follows: β3xo 0 ng, 7.6%; 0.5 ng, 55.7%; 2.5 ng, 96.0%. Data are indicated as mean ± SE (A and B, n = 5 from 1 frog; C, n = 5–6 from 2–3 frogs). Asterisks denote significant difference versus the control group without β3 (*P < 0.05, ***P < 0.001).
	Figure 5. . The α2δ subunit was not essential for the β3 subunit–induced inhibition of the N-type calcium channels. Oocytes were injected with cRNAs encoding Cav2.2α1 (2.5 ng/cell) in combination with various concentrations of β3. (A) I-V relationships for the peak Ba2+ currents at HPs of −80 (open circle) and −120 mV (closed circle). (B) Effects of the β3 subunit on the Gmax at different HPs. Gmax ratio (hatched column) was derived from Gmax at −80 (open column) and −120 mV (closed column). (C) Effect of the β3 subunit on steady-state inactivation. Inactivation midpoints (V1/2, inact) were calculated as follows: β3 0 ng (LVI and HVI), −66.7 and −49.8 mV; 2.5 ng, −84.2 and −48.6 mV; 12.5 ng, −85.7 and −44.3 mV. Proportions of LVI were as follows: β3 0 ng, 11.0%; 2.5 ng, 54.0%; 12.5 ng, 94.2%. Data are indicated as mean ± SE (A and B, n = 11 from 2 frogs; C, n = 4–8 from 2–4 frogs). Asterisks denote significant difference versus the control group without β3 (***P < 0.001).
	Figure 6. . The α2δ subunit markedly enhanced Gmax of the N-type calcium channels but had little or no effect on Gmax ratio. Oocytes were injected with cRNAs encoding Cav2.2α1 (2.5 ng/cell) in combination with α2δ (0, 2.5, or 12.5 ng/cell) in the presence (A and B) or absence (C and D) of the β3 subunit (2.5 ng/cell). (A and C) I-V relationships for the peak Ba2+ currents at HPs of −80 mV (open circle) and −120 mV (closed circle). (B and D) Effects of the α2δ subunit on Gmax at HPs of −80 (open column) and −120 mV (closed column) and Gmax ratio (hatched column). Data are indicated as mean ± SE (A and B, n = 5–6; C and D, n = 6 from 1 frog each). Asterisks denote significant difference versus the control group without β3 (*P < 0.05, **P < 0.01, ***P < 0.001).
	Figure 7. . Effects of the β3 subunit on R- (A–C) and L- (D–F) type calcium channels. For R-type channels, oocytes were injected with cRNA (cDNA for α1) encoding Cav2.3α1 (4.5 ng/cell) and α2δ (2.5 ng/cell) in combination with various concentrations of β3 (0, 0.5, 2.5 or 12.5 ng/cell). Cells expressing L-type channels were injected with cRNA encoding Cav1.2α1 (5 ng/cell), α2δ (12.5 ng/cell), and β3 (0, 5, or 25 ng/cell). (A and D) I-V relationships for the peak Ba2+ currents at HPs of −80 mV (open circle) and −120 mV (closed circle). (B and E) Effects of the β3 subunit on Gmax at different HPs. Gmax ratio (hatched column) was calculated from Gmax at −80 (open column) and −120 mV (closed column). (C and F) Effect of the β3 subunit on steady-state inactivation. Inactivation midpoints (V1/2, inact) were as follows: R-type/β3 0 ng (LVI and HVI), −94.7 and −63.3 mV; 2.5 ng, −89.5 and −64.7 mV; 12.5 ng, −86.5 and −58.5 mV; L-type/β3 0 ng (LVI and HVI), −65.3 and −35.0 mV; 5 ng, −67.1 and −32.8 mV; 25 ng, −64.2 and −33.0 mV. Proportion of LVI was as follows: R-type/β3 0 ng, 10.6%; 2.5 ng, 28.3%; 12.5 ng, 90.8%; L-type/β3 0 ng, 38.8%; 5 ng, 31.9%; 25 ng, 28.7%. Data are indicated as mean ± SE (A and B, n = 8 from 2 frogs; C, n = 4–7 from 2 frogs; D and E, n = 5 from 1 frog; F, n = 4 from 1 frog). Asterisks denote significant difference versus the control group without β3 (*P < 0.05, ***P < 0.001).
	Figure 8. . Slow kinetics of the closed-state inactivation of N- and R-type calcium channels. Oocytes were injected with cRNAs (cDNA for Cav2.3α1) encoding either Cav2.2α1 (2.5 ng/cell) or Cav2.3α1 (4.5 ng/cell) and α2δ (2.5 ng/cell) in the absence or presence of β3 (12.5 ng/cell). The kinetics of the inactivation and the recovery from the inactivation were investigated by altering HPs as indicated in each panel (A–D). Kinetic curves were derived from peak currents elicited by repetitive test pulses (100 ms) to 0 mV every 20 s as shown in the top. (A and B) Inactivation kinetics of N-type calcium channels. Cav2.2α1 with (A) or without β3 (B) corresponds to LVI or HVI of the steady-state inactivation curve of N-type channels, respectively (see Fig. 3 A). Closed diamonds in A are inactivation kinetics when channel activity was tested every 10 min instead of at 20-s intervals (n = 4). Inserts in B are control experiments indicating stable channel activity during a whole experiment (n = 8). (C and D) Inactivation kinetics of R-type calcium channels. Cav2.3α1 with (C) or without β3 (D) corresponds to LVI or HVI of the steady-state inactivation curve of R-type channels, respectively (see Fig. 7 C). All the inactivation and recovery kinetic curves were fitted statistically better to a two-phase exponential than to a mono exponential function (F-test). (E) Time constants (τ) and corresponding fractions for the kinetics of inactivation and recovery from the inactivation. F and S indicate a fast and a slow component, respectively. Data are indicated as mean ± SE (n = 4–6 from 2 frogs). (F) Steady-state inactivation with 30-min HPs. Broken lines show inactivation curves obtained with 3-min HPs for comparison (see Fig. 3 A). Each data point was obtained from individual oocytes and represents mean ± SE (n = 4–5 from 2–3 frogs).
	Figure 9. . Effects of different charge carriers (divalent cations) on the β3 subunit-induced inactivation of N-type calcium channels. Oocytes were injected with cRNAs encoding Cav2.2α1 (2.5 ng/cell), α2δ (2.5 ng/cell) and β3 (12.5 ng/cell). Recording was performed with a bath solution containing 5 or 40 mM Ba2+, or 5 mM Ca2+ as the charge carrier. (A) I-V relationships for the peak divalent cation currents at HPs of −80 (open circle) and −120 mV (closed circle). (B) Effects of the different charge carriers on Gmax at HPs of −80 (open column) and −120 mV (closed column) and Gmax ratio (hatched column). (C) Effects of the different charge carriers on steady-state inactivation. For simple comparison, inactivation midpoints (V1/2, inact) obtained with and without β3 were derived from a single Boltzmann equation: 5 mM Ba2+ (−β3 and +β3), −47.3 and −85.4 mV; 5 mM Ca2+, −39.1 and −81.0 mV; 40 mM Ba2+, −35.5 and −62.0 mV. Data are indicated as mean ± SE (A and B, n = 7 from 2 frogs; C, n = 5–6 from 2–4 frogs).

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