Functional Modularity of the β-Subunit of Voltage-Gated Ca2+ Channels Lin-ling He, Yun Zhang, Yu-hang Chen, Yoichi Yamada, Jian Yang  Biophysical Journal  Volume 93, Issue 3, Pages 834-845 (August 2007) DOI: 10.1529/biophysj.106.101691 Copyright © 2007 The Biophysical Society Terms and Conditions

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 Cav2.1 (E) or Cav1.2 (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 Cav1.2 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 Cav1.2. Middle column: HA staining under nonpermeablized conditions, illustrating Cav1.2 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 Cav1.2 instead of Cav2.1 for technical reasons (see Materials and Methods). (H) Average of HA staining intensity (in artificial unit) of Cav1.2 at the plasma membrane in the above three groups of HEK 293 cells. (I) Whole-oocyte Ca2+ channel peak current from oocytes expressing Cav2.1, 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 Cav1.2, α2δ, and either WT β3 or β3_GK (n=7–9). Untransfected cells and cells transfected with Cav1.2 and α2δ only had no currents. Biophysical Journal 2007 93, 834-845DOI: (10.1529/biophysj.106.101691) Copyright © 2007 The Biophysical Society Terms and Conditions

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 −20mV 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 +30mV (pulse A) was followed sequentially by a 25-s conditioning pulse (ranging from −60mV to +40mV) and a 20-ms test pulse to +30mV (pulse B). The holding potential was −80mV, and the interval between each protocol was 2min. 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 +30mV 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. Biophysical Journal 2007 93, 834-845DOI: (10.1529/biophysj.106.101691) Copyright © 2007 The Biophysical Society Terms and Conditions

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 45mM BaCl2, 80mM KCl, and 10mM HEPES (pH=7.3 with KOH) in the pipette. Biophysical Journal 2007 93, 834-845DOI: (10.1529/biophysj.106.101691) Copyright © 2007 The Biophysical Society Terms and Conditions

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. Biophysical Journal 2007 93, 834-845DOI: (10.1529/biophysj.106.101691) Copyright © 2007 The Biophysical Society Terms and Conditions

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. β2a_core/β1b_SH3 represents β2a core with the β1b SH3 domain, and β1b_core/β2a_SH3 represents β1b core with the β2a 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. β2a_core/β1b_HOOK presents β2a core with the β1b HOOK region, and β1b_core/β2a_HOOK represents β1b core with the β2a 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. Biophysical Journal 2007 93, 834-845DOI: (10.1529/biophysj.106.101691) Copyright © 2007 The Biophysical Society Terms and Conditions