J Gen Physiol
November 1, 2006;
Dose-dependent and isoform-specific modulation of Ca2+ channels by RGK GTPases.
Although inhibition of voltage-gated calcium channels by RGK GTPases (RGKs) represents an important mode of regulation to control Ca(2+) influx in excitable cells, their exact mechanism of inhibition remains controversial. This has prevented an understanding of how RGK regulation can be significant in a physiological context. Here we show that RGKs-Gem
, and Rem2
-decreased Ca(V)1.2 Ca(2+) current amplitude in a dose-dependent manner. Moreover, Rem2
, but not Rem
, produced dose-dependent alterations on gating kinetics, uncovering a new mode by which certain RGKs can precisely modulate Ca(2+) currents and affect Ca(2+) influx during action potentials. To explore how RGKs influence gating kinetics, we separated the roles mediated by the Ca(2+) channel accessory beta subunit''s interaction with its high affinity binding site in the pore-forming alpha(1C) subunit (AID) from its other putative contact sites by utilizing an alpha(1C)*beta3 concatemer in which the AID was mutated to prevent beta subunit interaction. This mutant concatemer generated currents with all the hallmarks of beta subunit modulation, demonstrating that AID-beta-independent interactions are sufficient for beta subunit modulation. Using this construct we found that although inhibition of current amplitude was still partially sensitive to RGKs, Rem2
no longer altered gating kinetics, implicating different determinants for this specific mode of Rem2
-mediated regulation. Together, these results offer new insights into the molecular mechanism of RGK-mediated Ca(2+) channel current modulation.
J Gen Physiol
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Figure 1. Dose-dependent inhibition of CaV1.2 channels by Gem and Rem2. (A) Normalized I-V relationships of IBa from oocytes injected with α1C/β3/α2δ without or with Gem (130 pg) or Rem2 (936 pg). n = 5. (B) Immunoblots with anti-Gem antibody. On left is shown GST and GST-Gem, demonstrating specificity of the antibody. The right panel shows an increase in the amount of Gem protein detected with an increasing amount (as indicated) of cRNA injected per oocyte. Gem is indicated by an arrow; a nonspecific band seen even without injection of Gem cRNA is shown by an asterisk. (C) Dose–response of Gem-, Rem-, or Rem2-mediated inhibition of IBa. KChIP2b cRNA was injected as a negative control. n = 5–7. (D) Exemplar traces of CaV1.2 channels expressed without a RGK, with Gem (20 pg), with Rem (100 pg), or with Rem2 (468 pg). Bars: 1 s and 1 μA.
Figure 2. Rem2 affects channel activation and inactivation. (A) Top panels show exemplar current traces during a 2-s test pulse at +10 mV for Gem and Rem2 at the indicated doses. Bars, 1 μA. The bottom panels show scaled exemplar traces of α1C/β3/α2δ (gray) coinjected with Gem (65 pg) or Rem2 (936 pg) (black) during a 2-s test pulse to +10 mV. (B) Steady-state inactivation for α1C/β3/α2δ without or with Gem (26 pg) or Rem2 (936 pg). n = 10–12 (C–E) Dose–response of Rem2-mediated effects upon kinetics of activation (τfast, fraction Aslow, and τslow, respectively, at the indicated test potentials—see legend in C—and with the amounts of cRNA injected as indicated on the x-axis), n = 7–14. (F) Scaled exemplar traces of activation phases at +10 mV for α1C/β3/α2δ coexpressed with the indicated Rem2 doses. Bar, 10 ms. (G) Ca2+ currents recorded during a simulated burst of action potentials in a pancreatic β cell (see Materials and methods) with no RGK or Rem2 (468 pg). Bars: 200 nA, 1 s.
Figure 3. The Ca2+ channel β subunit, but not the α1C N and C termini, are required for Gem- or Rem2-mediated effects. (A and B) Normalized IBa for the indicated combinations of α1C (WT), the N-terminal truncation (ΔN), or the C-terminal truncation (ΔC), with or with Gem or Rem2, as indicated. All combinations were coexpressed with β3 and α2δ. Scaled exemplar current for each pair with (black) or without (gray) Gem or Rem2 are shown on right. n = 5–9. Bars: 50 ms (A) and 1 s (B). (C and D) Normalized IBa for the indicated combinations of ΔC and α2δ with or without β3 and Gem or Rem2, as indicated. For the pair without β3, scaled exemplar currents with (black) or without (gray) Gem or Rem2 are shown on right. n = 5. Scale bars as above.
Figure 4. Interaction between the Ca2+ channel β subunits and the AID influence Gem- and Rem2-mediated effects. (A) Normalized I-V relationships of IBa from oocytes injected with the indicated constructs or combinations, all coinjected with α2δ. n = 31–34. (B) A Hisx6 immunoblot of a pull-down experiment for purified β2 SH3-GK core (Maltez et al., 2005) using a GST-α1C I-II or I-IIYW mutant loop. Coomassie-stained gel below shows equal loading of the GST fusion proteins. (C) Exemplar traces and models of the indicated concatemers or combinations. Bars: 1 s, 4 μA. (D–F) Normalized IBa for the indicated concatemers or combinations (all expressed with α2δ), with or without Gem or Rem2. Scaled exemplar traces are shown on right. n = 5. Bars: 1 s (D and F) and 50 ms (E).
Figure 5. Rem2 has extended termini. CLUSTAL W (1.83) multiple sequence alignment (Thompson et al., 1994) of Rem, Gem, and Rem2. The gray-boxed areas highlight the extended N and C termini in Rem2 that flank the conserved Ras-like core.
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