Auxiliary subunits operate as a molecular switch in determining gating behaviour of the unitary N-type Ca2+ channel current in Xenopus oocytes

Abstract

1. We systematically examined the biophysical properties of omega-conotoxin GVIA-sensitive neuronal N-type channels composed of various combinations of the alpha1B, alpha2/delta and beta1b subunits in Xenopus oocytes. 2. Whole-cell recordings demonstrated that coexpression of the beta1b subunit decelerated inactivation, whereas the alpha2/delta accelerated both activation and inactivation, and cancelled the kinetic effects of the beta1b. The alpha2/delta and the beta1b controlled voltage dependence of activation differently: the beta1b significantly shifted the current-voltage relationship towards the hyperpolarizing direction; however, the alpha2/delta shifted the relationship only slightly in the depolarizing direction. The extent of voltage-dependent inactivation was modified solely by the beta1b. 3. Unitary currents measured using a cell-attached patch showed stable patterns of opening that were markedly different among subunit combinations in their kinetic parameters. The alpha2/delta and the beta1b subunits also acted antagonistically in regulating gating patterns of unitary N-type channels. Open time was shortened by the alpha2/delta, while the fraction of long opening was enhanced by the beta1b. The alpha2/delta decreased opening probability (Po), while the beta1b increased Po. alpha1Balpha2/deltabeta1b produced unitary activity with an open time distribution value in between those of alpha1Balpha2/delta and alpha1Bbeta1b. However, both the alpha2/delta and the beta1b subunits reduced the number of null traces. 4. These results suggest that the auxiliary subunits alone and in combination contribute differently in forming gating apparatuses in the N-type channel, raising the possibility that subunit interaction contributes to the generation of functional diversity of N-type channels in native neuronal preparations also.

Figure 7. Kinetic analysis of open and closed times for the N-type channels composed of various subunit combinations

Open and closed time distributions of the channels for α_1B alone (A), α_1Bα₂/δ (B), α_1Bβ_1b (C) and α_1Bα₂/δβ_1b (D) were plotted from the single-channel recording in a cell-attached configuration shown in Fig. 6. The open times of each N-type channel are fitted by a single exponential function for α_1Bα₂/δ and the sum of two exponential functions for the other three subunit combinations. The closed times of each N-type channel are fitted by the sum of two exponential functions. The limitation of the fit is set at 25 ms. Numbers in the histograms represents open or closed times and their relative area is in parentheses. Subscripts o, c, f and s for the time constants represent open, closed, fast and slow, respectively.

Figure 1. Pharmacological properties of the recombinant N-type channels

A, effects of Ca²⁺ channel antagonists and toxins on recombinant N-type channels expressed with the α_1B, the α₂/δ and the β_1b subunits in Xenopus oocytes. Examples of Ba²⁺ currents elicited by 115 ms depolarizing pulses to 10 mV from a holding potential (V_h) of −100 mV before and during exposure to 10 μm Cd²⁺ (a), 1 μm (−)Bay K 8644 (b), 200 nmω-Aga IVA (c) and 1 μmω-CgTx GVIA (d). Ba²⁺ currents were recorded just before (control) and 3 min after application of each agent. External Ba²⁺ concentration was 4 mM. Cytochrome C (0.1 mg ml⁻¹), to saturate non-specific peptide binding, was added to the external solution in the experiments of c and d. B, inhibition of N-type channels, composed of various subunit combinations, by ω-CgTx GVIA. a, oocyte was treated with 1 μmω-CgTx GVIA in the external solution containing 4 mM Ba²⁺ for 3 min between the records measured in 40 mM Ba²⁺ before application and after washing out of ω-CgTx GVIA. Currents were elicited by 250 ms step pulses to 20 mV from a V_h of −100 mV. b and c, oocyte was treated with 1 μmω-CgTx GVIA in the external solution containing 4 mM Ba²⁺ for 3 min. Step depolarizations were applied to 10 mV (b) and 0 mV (c) from a V_h of −100 mV in the external solution containing 4 mM Ba²⁺. Small capacity currents remaining after subtraction have been blanked.

Figure 2. Effects of the α₂/δ and the β_1b subunits on the macroscopic α_1B currents

The α_1B channels were expressed alone (A) or together with the α₂/δ subunit (B), the β_1b subunit (C), or both the α₂/δ and the β_1b subunits (D). Ba²⁺ currents, which were evoked with 40 mM external Ba²⁺ by step depolarizations to voltages between −20 and 50 mV (A, C and D) and between −10 and 60 mV (B) in 10 mV intervals from a V_h of −100 mV, are shown on slow (left panel) and fast (middle panel) scales. Small capacity currents remaining after subtraction have been blanked. The corresponding peak current-voltage relationships are shown in the right panel. Curves were drawn by an interpolation process.

Figure 3. The β_1b but not the α₂/δ subunit changes the voltage-dependent inactivation properties of the α_1B channels

Voltage-dependent inactivation of the α_1B channels expressed alone or together with the α₂/δ subunit and/or the β_1b subunit is compared. A, voltage-dependent inactivation was studied using a conventional double-pulse protocol. a, inactivation was induced by 5 s potential displacements (conditioning pulse) from −100 to 40 mV with increments of 10 mV immediately before 200 ms test pulses to 20 mV. b and c, Ba²⁺ currents were evoked by test pulses to 20 mV followed by 5 s conditioning pulses at the indicated potential in oocytes injected with α_1Bα₂/δ (b) and α_1Bβ_1b (c). B, the amplitudes of Ba²⁺ currents elicited by test pulses after conditioning pulses were normalized to the amplitude at a conditioning pulse potential of −100 mV and plotted against the conditioning potential. Data points were fitted with a smooth curve derived from the Boltzmann equation I/I_max= (1 + exp(V_m - V_0.5)/k)⁻¹ where V_m is prepulse potential, V_0.5 is half-inactivation potential, and k is a slope factor. The values of V_0.5 and k are −10.9 mV and 7.34 mV for α_1B alone (○), −13.4 mV and 6.74 mV for α_1Bα₂/δ (•), −35.2 mV and 15.86 mV for α_1Bβ_1b (▵), and −33.4 mV and 12.44 mV for α_1Bα₂/δβ_1b (▴), respectively. Vertical bars show means ±s.e.m. of 5 measurements if they are larger than symbols.

Figure 4. Influence of the α₂/δ and the β_1b subunits on activation (time to peak) (A) and inactivation (time constant) (B) kinetics

The α_1B channels were expressed alone or together with the α₂/δ subunit and/or β_1b subunit as indicated. The experimental conditions used were the same as in Fig. 2. Data points represent means ±s.e.m. of 6–17 measurements. ○, α_1B alone; •, α_1Bα₂/δ; ▵, α_1Bβ_1b; and ▴, α_1Bα₂/δβ_1b. Vertical bars show means ±s.e.m. if they are larger than symbols.

Figure 5. Unitary current-voltage relationship for the recombinant N-type channel expressed with the α_1B, the α₂/δ and the β_1b subunits in Xenopus oocytes

A, three consecutive representative sweeps, recorded in a cell-attached configuration from oocytes injected with cRNA specific to α_1B alone (a), α_1Bα₂/δ (b), α_1Bβ_1b (c) or α_1Bα₂/δβ_1b (d), are shown. The indicated test depolarization of 150 ms duration was applied every 3 s from a V_h of −100 mV. The pipette solution contained 110 mM Ba²⁺. Arrowheads indicate when the test depolarizations began and ended. B, unitary current-voltage relationship. Each point represents mean ±s.e.m. of 5–7 patches. Data were fitted by a linear regression with slopes of 16.3, 16.6, 15.6 and 17.3 pS for α_1B alone (○), α_1Bα₂/δ (•), α_1Bβ_1b (▵) and α_1Bα₂/δβ_1b (▴), respectively. Data points represent the mean of 5–8 measurements. The inset shows the amplitude histogram for the data illustrated in Ad. The test depolarization was 10 mV.

Figure 6. Different unitary activity of the recombinant N-type channel expressed with the α_1B, the α₂/δ and the β_1b subunits

Unitary activity of the N-type channel, expressed with α_1B subunit alone (A), α_1Bα₂/δ (B), α_1Bβ_1b (C) and α_1Bα₂/δβ_1b (D), were recorded in a cell-attached configuration. The step pulses to 20 mV for 150 ms were applied every 3 s from a V_h of −100 mV. Sixteen consecutive, leak-subtracted current traces are shown. The experimental conditions used were the same as in Fig. 5 unless specified otherwise.

Figure 8. Ensemble average currents of the unitary N-type channels and their curve fitting

Ensemble currents, which were evoked every 5 s by 450 ms step depolarization to 40 mV from a V_h of −100 mV, are the average of 100 for α_1B alone (A), 100 for α_1Bα₂/δ (B), 75 for α_1Bβ_1b (C) and 50 individual sweeps for α_1Bα₂/δβ_1b (D). Recording electrodes contained at least 2, 4, 3 and 3 channels for α_1B alone, α_1Bα₂/δ, α_1Bβ_1b and α_1Bα₂/δβ_1b, respectively. Decay components of the ensemble average current are fitted by a single exponential function with a time constant of 146.1 ms (A), 96.9 ms (B), 335.4 ms (C) and 140.0 ms (D).