Functional modularity of the beta-subunit of voltage-gated Ca2+ channels

Abstract

The beta-subunit of voltage-gated Ca(2+) channels plays a dual role in chaperoning the channels to the plasma membrane and modulating their gating. It contains five distinct modular domains/regions, including the variable N- and C-terminus, a conserved Src homology 3 (SH3) domain, a conserved guanylate kinase (GK) domain, and a connecting variable and flexible HOOK region. Recent crystallographic studies revealed a highly conserved interaction between the GK domain and alpha interaction domain (AID), the high-affinity binding site in the pore-forming alpha(1) subunit. Here we show that the AID-GK domain interaction is necessary for beta-subunit-stimulated Ca(2+) channel surface expression and that the GK domain alone can carry out this function. We also examined the role of each region of all four beta-subunit subfamilies in modulating P/Q-type Ca(2+) channel gating and demonstrate that the beta-subunit functions modularly. Our results support a model that the conserved AID-GK domain interaction anchors the beta-subunit to the alpha(1) subunit, enabling alpha(1)-beta pair-specific low-affinity interactions involving the N-terminus and the HOOK region, which confer on each of the four beta-subunit subfamilies its distinctive modulatory properties.

FIGURE 1

The AID-GK domain interaction is essential and sufficient for Ca²⁺ channel surface expression. (A) Schematic domain organization of Ca²⁺ channel β-subunits. (B) Close-up of the AID-GK domain interaction interface of the β3 subunit. (C) List of mutations in the β₃ subunit. (D) Coomassie blue staining illustrating the interaction between the indicated WT or mutant β₃ cores and the AID. GST-AID was immobilized in a GST column and was used to pull down the various β₃ cores. b: bound; u: unbound. (E, F) Whole-oocyte Ca²⁺ channel peak current from oocytes expressing Ca_v2.1 (E) or Ca_v1.2 (F), α₂δ and the indicated WT or mutant β₃ (β⁻: no β-subunit was injected). (G) Confocal images of nonpermeabilized HEK 293 cells transfected with Ca_v1.2 subunit tagged with GFP and HA, either alone or with WT β₃ or a mutant β₃ subunit (mutant 1). Left column: GFP fluorescence illustrating the distribution of Ca_v1.2. Middle column: HA staining under nonpermeablized conditions, illustrating Ca_v1.2 on the plasma membrane, superimposed with the brightfield image of the cells. Right column: intensity of HA staining (in artificial unit) at the plasma membrane at points indicated in the middle column. We used Ca_v1.2 instead of Ca_v2.1 for technical reasons (see Materials and Methods). (H) Average of HA staining intensity (in artificial unit) of Ca_v1.2 at the plasma membrane in the above three groups of HEK 293 cells. (I) Whole-oocyte Ca²⁺ channel peak current from oocytes expressing Ca_v2.1, α₂δ, and either WT β₃ or β₃ GK domain (β₃_GK). (J) Current-voltage relationship of whole-cell Ca²⁺ channel currents from HEK 293T cells transfected with Ca_v1.2, α₂δ, and either WT β₃ or β₃_GK (n = 7–9). Untransfected cells and cells transfected with Ca_v1.2 and α₂δ only had no currents.

FIGURE 2

Functional effects of the GK domain. (A) Voltage dependence of activation of β⁻ channels and channels containing the indicated GK domain. In this and the following figures, data points represent normalized tail currents recorded at −20 mV after depolarization to a given test potential. N = 5–14. (B) Voltage dependence of steady-state inactivation of channels containing the indicated GK domain. In this and the following figures, steady-state inactivation was determined by a three-pulse protocol in which a 20-ms normalizing pulse to +30 mV (pulse A) was followed sequentially by a 25-s conditioning pulse (ranging from −60 mV to +40 mV) and a 20-ms test pulse to +30 mV (pulse B). The holding potential was −80 mV, and the interval between each protocol was 2 min. Peak current evoked by pulse B was normalized by that evoked by pulse A and was plotted against the conditioning potentials. N = 5–7. (C) Comparison of the kinetics of inactivation of channels containing the indicated GK domain. In this and the following figures, current was evoked by the 25-s conditioning depolarization to +30 mV from above and was normalized by the peak amplitude. (D–G) Comparison of the voltage dependence of steady-state inactivation of channels containing the indicated WT or mutant β-subunit. N = 5–11. (H–K) Comparison of the kinetics of inactivation of channels containing the indicated WT β-subunit or GK domain. Currents were recorded in inside-out patches.

FIGURE 3

Deletion of the β-subunit C-terminus has no functional effects. (A–D) Comparison of the voltage dependence of activation of channels containing the indicated WT or mutant β-subunit. N = 5–10. (E–H) Comparison of the voltage dependence of steady-state inactivation of channels containing the indicated WT or mutant β-subunit. N = 5–9. (I–L) Comparison of the kinetics of inactivation of channels containing the indicated WT or mutant β-subunit. Black and gray lines represent WT and mutant β-subunits, respectively. These experiments were performed in cell-attached macropatch recordings with 45 mM BaCl₂, 80 mM KCl, and 10 mM HEPES (pH = 7.3 with KOH) in the pipette.

FIGURE 4

Functional effects of the β core domain. (A–D) Comparison of the voltage dependence of activation of channels containing the indicated WT β-subunit or β core. N = 5–11. (E–H) Comparison of the voltage dependence of steady-state inactivation of channels containing the indicated WT β-subunit or β core. N = 4–7. (I–L) Comparison of the kinetics of inactivation of channels containing the indicated WT β-subunit or β core. (M) Comparison of the kinetics of inactivation of channels containing the indicated β core. Gray line represents β⁻ channels. Currents were recorded in inside-out patches.

FIGURE 5

Functional effects of the SH3 domain and HOOK region. (A) Comparison of the voltage dependence of steady-state inactivation of channels containing the indicated β-subunit mutant. β_2a_core/β_1b_SH3 represents β_2a core with the β_1b SH3 domain, and β_1b_core/β_2a_SH3 represents β_1b core with the β_2a SH3 domain. N = 5–6. (B) Comparison of the kinetics of inactivation of channels containing the indicated β-subunit mutant. (C) Comparison of the voltage dependence of activation of channels containing the indicated β-subunit mutant. N = 5–8. (D) Comparison of the voltage dependence of steady-state inactivation of channels containing the indicated β-subunit mutant. β_2a_core/β_1b_HOOK presents β_2a core with the β_1b HOOK region, and β_1b_core/β_2a_HOOK represents β_1b core with the β_2a HOOK region. N = 4–6. (E) Comparison of the kinetics of inactivation of channels containing the indicated β-subunit mutant. (F) Comparison of the voltage dependence of activation of channels containing the indicated β-subunit mutant. N = 6–7. Currents were recorded in inside-out patches.