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J Gen Physiol
2006 Nov 01;1285:605-13. doi: 10.1085/jgp.200609631.
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Dose-dependent and isoform-specific modulation of Ca2+ channels by RGK GTPases.
Seu L
,
Pitt GS
.
Abstract
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, Rem, and Rem2-decreased Ca(V)1.2 Ca(2+) current amplitude in a dose-dependent manner. Moreover, Rem2, but not Rem or Gem, 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.
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.
An,
Modulation of A-type potassium channels by a family of calcium sensors.
2000, Pubmed,
Xenbase
An,
Modulation of A-type potassium channels by a family of calcium sensors.
2000,
Pubmed
,
Xenbase
Budde,
Calcium-dependent inactivation of neuronal calcium channels.
2002,
Pubmed
Béguin,
Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem.
2001,
Pubmed
,
Xenbase
Béguin,
14-3-3 and calmodulin control subcellular distribution of Kir/Gem and its regulation of cell shape and calcium channel activity.
2005,
Pubmed
Béguin,
Roles of 14-3-3 and calmodulin binding in subcellular localization and function of the small G-protein Rem2.
2005,
Pubmed
Cantí,
Evidence for two concentration-dependent processes for beta-subunit effects on alpha1B calcium channels.
2001,
Pubmed
,
Xenbase
Catterall,
Structure and regulation of voltage-gated Ca2+ channels.
2000,
Pubmed
Chen,
Expression of Rem2, an RGK family small GTPase, reduces N-type calcium current without affecting channel surface density.
2005,
Pubmed
Crump,
L-type calcium channel alpha-subunit and protein kinase inhibitors modulate Rem-mediated regulation of current.
2006,
Pubmed
Dalton,
A single CaVbeta can reconstitute both trafficking and macroscopic conductance of voltage-dependent calcium channels.
2005,
Pubmed
Dolphin,
Beta subunits of voltage-gated calcium channels.
2003,
Pubmed
Finlin,
Regulation of L-type Ca2+ channel activity and insulin secretion by the Rem2 GTPase.
2005,
Pubmed
Finlin,
Analysis of the complex between Ca2+ channel beta-subunit and the Rem GTPase.
2006,
Pubmed
Finlin,
Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases.
2000,
Pubmed
Finlin,
Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases.
2003,
Pubmed
Finlin,
Rem is a new member of the Rad- and Gem/Kir Ras-related GTP-binding protein family repressed by lipopolysaccharide stimulation.
1997,
Pubmed
Gerhardstein,
Proteolytic processing of the C terminus of the alpha(1C) subunit of L-type calcium channels and the role of a proline-rich domain in membrane tethering of proteolytic fragments.
2000,
Pubmed
Ivanina,
Modulation of L-type Ca2+ channels by gbeta gamma and calmodulin via interactions with N and C termini of alpha 1C.
2000,
Pubmed
Juntti-Berggren,
Increased activity of L-type Ca2+ channels exposed to serum from patients with type I diabetes.
1993,
Pubmed
Kanno,
Glucose-dependent regulation of rhythmic action potential firing in pancreatic beta-cells by K(ATP)-channel modulation.
2002,
Pubmed
Kelly,
The RGK family: a regulatory tail of small GTP-binding proteins.
2005,
Pubmed
Kim,
Identification of the components controlling inactivation of voltage-gated Ca2+ channels.
2004,
Pubmed
,
Xenbase
Klöckner,
Involvement of the carboxyl-terminal region of the alpha 1 subunit in voltage-dependent inactivation of cardiac calcium channels.
1995,
Pubmed
Leone,
The Gem GTP-binding protein promotes morphological differentiation in neuroblastoma.
2001,
Pubmed
Leroy,
Interaction via a key tryptophan in the I-II linker of N-type calcium channels is required for beta1 but not for palmitoylated beta2, implicating an additional binding site in the regulation of channel voltage-dependent properties.
2005,
Pubmed
Maguire,
Gem: an induced, immediate early protein belonging to the Ras family.
1994,
Pubmed
Maltez,
Essential Ca(V)beta modulatory properties are AID-independent.
2005,
Pubmed
,
Xenbase
Mears,
Regulation of insulin secretion in islets of Langerhans by Ca(2+)channels.
2004,
Pubmed
Murata,
Creation of a genetic calcium channel blocker by targeted gem gene transfer in the heart.
2004,
Pubmed
Ohsugi,
Glucose and insulin treatment of insulinoma cells results in transcriptional regulation of a common set of genes.
2004,
Pubmed
Pragnell,
Calcium channel beta-subunit binds to a conserved motif in the I-II cytoplasmic linker of the alpha 1-subunit.
1994,
Pubmed
Reynet,
Rad: a member of the Ras family overexpressed in muscle of type II diabetic humans.
1993,
Pubmed
Sasaki,
Direct inhibition of the interaction between alpha-interaction domain and beta-interaction domain of voltage-dependent Ca2+ channels by Gem.
2005,
Pubmed
Singer,
The roles of the subunits in the function of the calcium channel.
1991,
Pubmed
,
Xenbase
Splawski,
Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism.
2004,
Pubmed
,
Xenbase
Takahashi,
A CaVbeta SH3/guanylate kinase domain interaction regulates multiple properties of voltage-gated Ca2+ channels.
2005,
Pubmed
Thompson,
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
1994,
Pubmed
Van Petegem,
Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain.
2004,
Pubmed
Walker,
A beta 4 isoform-specific interaction site in the carboxyl-terminal region of the voltage-dependent Ca2+ channel alpha 1A subunit.
1998,
Pubmed
Ward,
The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway.
2002,
Pubmed
Ward,
Phosphorylation of critical serine residues in Gem separates cytoskeletal reorganization from down-regulation of calcium channel activity.
2004,
Pubmed
Wei,
Modification of Ca2+ channel activity by deletions at the carboxyl terminus of the cardiac alpha 1 subunit.
1994,
Pubmed
,
Xenbase
Zühlke,
Ca2+-sensitive inactivation and facilitation of L-type Ca2+ channels both depend on specific amino acid residues in a consensus calmodulin-binding motif in the(alpha)1C subunit.
2000,
Pubmed
