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A CaVbeta SH3/guanylate kinase domain interaction regulates multiple properties of voltage-gated Ca2+ channels.

Takahashi SX, Miriyala J, Tay LH, Yue DT, Colecraft HM - J. Gen. Physiol. (2005)

Bottom Line: These effects are mediated through a characteristic src homology 3/guanylate kinase (SH3-GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins.A more extreme case, in which the trans SH3-GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the alpha(1C) (Ca(V)1.2) subunit.The results reveal that Ca(V)beta SH3 and GK domains function codependently to tune Ca(2+) channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca(2+) channel activity.

View Article: PubMed Central - PubMed

Affiliation: Calcium Signals Laboratory, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Auxiliary Ca(2+) channel beta subunits (Ca(V)beta) regulate cellular Ca(2+) signaling by trafficking pore-forming alpha(1) subunits to the membrane and normalizing channel gating. These effects are mediated through a characteristic src homology 3/guanylate kinase (SH3-GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins. However, the mechanisms by which the Ca(V)beta SH3-GK module regulates multiple Ca(2+) channel functions are not well understood. Here, using a split-domain approach, we investigated the role of the interrelationship between Ca(V)beta SH3 and GK domains in defining channel properties. The studies build upon a previously identified split-domain pair that displays a trans SH3-GK interaction, and fully reconstitutes Ca(V)beta effects on channel trafficking, activation gating, and increased open probability (P(o)). Here, by varying the precise locations used to separate SH3 and GK domains and monitoring subsequent SH3-GK interactions by fluorescence resonance energy transfer (FRET), we identified a particular split-domain pair that displayed a subtly altered configuration of the trans SH3-GK interaction. Remarkably, this pair discriminated between Ca(V)beta trafficking and gating properties: alpha(1C) targeting to the membrane was fully reconstituted, whereas shifts in activation gating and increased P(o) functions were selectively lost. A more extreme case, in which the trans SH3-GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the alpha(1C) (Ca(V)1.2) subunit. The results reveal that Ca(V)beta SH3 and GK domains function codependently to tune Ca(2+) channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca(2+) channel activity.

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Deletion of a SH3 β5 strand selectively ablates the CaVβ SH3–GK interaction. (A) Cartoon comparing structures of GKC and GKC[ΔPYDVV]. (B) Confocal slices showing subcellular localization of CFP-NSH3 and YFP-GKC[ΔPYDVV]. (C) Population FR and EEFF measurements for CaVβ SH3/GK domain interactions. (D) Confocal slices showing subcellular localization of CFP-α1C[I-II loop] and distinct YFP-tagged CaVβ GK domains. (E) Population FR and EEFF measurements for α1C[I-II loop]/GK domain interactions.
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fig5: Deletion of a SH3 β5 strand selectively ablates the CaVβ SH3–GK interaction. (A) Cartoon comparing structures of GKC and GKC[ΔPYDVV]. (B) Confocal slices showing subcellular localization of CFP-NSH3 and YFP-GKC[ΔPYDVV]. (C) Population FR and EEFF measurements for CaVβ SH3/GK domain interactions. (D) Confocal slices showing subcellular localization of CFP-α1C[I-II loop] and distinct YFP-tagged CaVβ GK domains. (E) Population FR and EEFF measurements for α1C[I-II loop]/GK domain interactions.

Mentions: To address this deficiency, we focused attention on a five-residue β5 strand (that occurs immediately after the variable V2 domain and is formally a part of the SH3 domain) that the crystal structures suggested is crucial for keeping the two domains together by interacting with residues in both the SH3 and GK domains (Opatowsky et al., 2003; Chen et al., 2004; Van Petegem et al., 2004). We hypothesized that deleting the β5 strand would selectively ablate the CaVβ SH3–GK interaction. To test this, we deleted the β5 strand from GKC, generating GKC[ΔPYDVV] (Fig. 5 A). YFP-GKC[ΔPYDVV] was diffusely localized throughout the cytosol and excluded from the nucleus (Fig. 5 B, right), similar to the subcellular localization of YFP-GKC (Fig. 4 A). Three-cube FRET experiments revealed that YFP-GKC[ΔPYDVV] did not interact with CFP-NSH3 (FR = 0.9 ± 0.07, n = 6) indicating a successful ablation of the SH3–GK interaction (Fig. 5 C), and affirming the critical role of the β5 strand in keeping the two domains together.


A CaVbeta SH3/guanylate kinase domain interaction regulates multiple properties of voltage-gated Ca2+ channels.

Takahashi SX, Miriyala J, Tay LH, Yue DT, Colecraft HM - J. Gen. Physiol. (2005)

Deletion of a SH3 β5 strand selectively ablates the CaVβ SH3–GK interaction. (A) Cartoon comparing structures of GKC and GKC[ΔPYDVV]. (B) Confocal slices showing subcellular localization of CFP-NSH3 and YFP-GKC[ΔPYDVV]. (C) Population FR and EEFF measurements for CaVβ SH3/GK domain interactions. (D) Confocal slices showing subcellular localization of CFP-α1C[I-II loop] and distinct YFP-tagged CaVβ GK domains. (E) Population FR and EEFF measurements for α1C[I-II loop]/GK domain interactions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266626&req=5

fig5: Deletion of a SH3 β5 strand selectively ablates the CaVβ SH3–GK interaction. (A) Cartoon comparing structures of GKC and GKC[ΔPYDVV]. (B) Confocal slices showing subcellular localization of CFP-NSH3 and YFP-GKC[ΔPYDVV]. (C) Population FR and EEFF measurements for CaVβ SH3/GK domain interactions. (D) Confocal slices showing subcellular localization of CFP-α1C[I-II loop] and distinct YFP-tagged CaVβ GK domains. (E) Population FR and EEFF measurements for α1C[I-II loop]/GK domain interactions.
Mentions: To address this deficiency, we focused attention on a five-residue β5 strand (that occurs immediately after the variable V2 domain and is formally a part of the SH3 domain) that the crystal structures suggested is crucial for keeping the two domains together by interacting with residues in both the SH3 and GK domains (Opatowsky et al., 2003; Chen et al., 2004; Van Petegem et al., 2004). We hypothesized that deleting the β5 strand would selectively ablate the CaVβ SH3–GK interaction. To test this, we deleted the β5 strand from GKC, generating GKC[ΔPYDVV] (Fig. 5 A). YFP-GKC[ΔPYDVV] was diffusely localized throughout the cytosol and excluded from the nucleus (Fig. 5 B, right), similar to the subcellular localization of YFP-GKC (Fig. 4 A). Three-cube FRET experiments revealed that YFP-GKC[ΔPYDVV] did not interact with CFP-NSH3 (FR = 0.9 ± 0.07, n = 6) indicating a successful ablation of the SH3–GK interaction (Fig. 5 C), and affirming the critical role of the β5 strand in keeping the two domains together.

Bottom Line: These effects are mediated through a characteristic src homology 3/guanylate kinase (SH3-GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins.A more extreme case, in which the trans SH3-GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the alpha(1C) (Ca(V)1.2) subunit.The results reveal that Ca(V)beta SH3 and GK domains function codependently to tune Ca(2+) channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca(2+) channel activity.

View Article: PubMed Central - PubMed

Affiliation: Calcium Signals Laboratory, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Auxiliary Ca(2+) channel beta subunits (Ca(V)beta) regulate cellular Ca(2+) signaling by trafficking pore-forming alpha(1) subunits to the membrane and normalizing channel gating. These effects are mediated through a characteristic src homology 3/guanylate kinase (SH3-GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins. However, the mechanisms by which the Ca(V)beta SH3-GK module regulates multiple Ca(2+) channel functions are not well understood. Here, using a split-domain approach, we investigated the role of the interrelationship between Ca(V)beta SH3 and GK domains in defining channel properties. The studies build upon a previously identified split-domain pair that displays a trans SH3-GK interaction, and fully reconstitutes Ca(V)beta effects on channel trafficking, activation gating, and increased open probability (P(o)). Here, by varying the precise locations used to separate SH3 and GK domains and monitoring subsequent SH3-GK interactions by fluorescence resonance energy transfer (FRET), we identified a particular split-domain pair that displayed a subtly altered configuration of the trans SH3-GK interaction. Remarkably, this pair discriminated between Ca(V)beta trafficking and gating properties: alpha(1C) targeting to the membrane was fully reconstituted, whereas shifts in activation gating and increased P(o) functions were selectively lost. A more extreme case, in which the trans SH3-GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the alpha(1C) (Ca(V)1.2) subunit. The results reveal that Ca(V)beta SH3 and GK domains function codependently to tune Ca(2+) channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca(2+) channel activity.

Show MeSH