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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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Probing CaVβ split-domain interactions by three-cube FRET. (A) Confocal images of a HEK 293 cell coexpressing CFP-NSH3 and YFP-GKC (top), or CFP-NSH3 and YFP-GKC[trunc] (bottom). (B) Mean fluorescent intensities for YFP-GKC (n = 25) and YFP-GKC[trunc] (n = 26) molecules measured in transfected HEK 293 cells. (C) Population FR and EEFF measurements. (D and E) Fits of FR scatter plots for CFP-NSH3+YFP-GKC and CFP-NSH3+YFP-GKC[trunc] to a 1:1 binding model.
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fig4: Probing CaVβ split-domain interactions by three-cube FRET. (A) Confocal images of a HEK 293 cell coexpressing CFP-NSH3 and YFP-GKC (top), or CFP-NSH3 and YFP-GKC[trunc] (bottom). (B) Mean fluorescent intensities for YFP-GKC (n = 25) and YFP-GKC[trunc] (n = 26) molecules measured in transfected HEK 293 cells. (C) Population FR and EEFF measurements. (D and E) Fits of FR scatter plots for CFP-NSH3+YFP-GKC and CFP-NSH3+YFP-GKC[trunc] to a 1:1 binding model.

Mentions: We adopted the optical FRET two-hybrid approach (Erickson et al., 2003) to probe for interactions between split-domain CaVβ constructs (Fig. 4). This approach offered the advantage of reporting on the status of split-domain CaVβ SH3–GK interactions in the relevant setting of live HEK 293 cells, permitting a direct correlation of binding properties and functional electrophysiological experiments. Coding sequences for enhanced CFP and enhanced YFP were fused upstream of those for NSH3 and the two GK domain constructs, respectively. Confocal images of live cells show the subcellular localization of the distinct XFP-tagged proteins upon their cotransfection in HEK 293 cells (Fig. 4 A). CFP-NSH3 localized uniformly throughout the cell, with similar fluorescent intensity in the cytoplasm and the nucleus (Fig. 4 A, left). This distribution presumably reflects the fact that the relatively small size of CFP-NSH3 permits passive diffusion into the nucleus. By contrast, both YFP-GKC and YFP-GKC[trunc] were uniformly present in the cytosol, and excluded from the nucleus (Fig. 4 A, right). Importantly, the mean fluorescence intensities for YFP-GKC and YFP-GKC-[trunc] were not significantly different (Fig. 4 B; P = 0.07), indicating no dramatic differences in expression levels between these two proteins. This finding rules out the trivial possibility that the observed functional divergence between GKC and GKC[trunc] (Figs. 2 and 3) could be due to large differences in expression levels or protein stability between the two.


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)

Probing CaVβ split-domain interactions by three-cube FRET. (A) Confocal images of a HEK 293 cell coexpressing CFP-NSH3 and YFP-GKC (top), or CFP-NSH3 and YFP-GKC[trunc] (bottom). (B) Mean fluorescent intensities for YFP-GKC (n = 25) and YFP-GKC[trunc] (n = 26) molecules measured in transfected HEK 293 cells. (C) Population FR and EEFF measurements. (D and E) Fits of FR scatter plots for CFP-NSH3+YFP-GKC and CFP-NSH3+YFP-GKC[trunc] to a 1:1 binding model.
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Related In: Results  -  Collection

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

fig4: Probing CaVβ split-domain interactions by three-cube FRET. (A) Confocal images of a HEK 293 cell coexpressing CFP-NSH3 and YFP-GKC (top), or CFP-NSH3 and YFP-GKC[trunc] (bottom). (B) Mean fluorescent intensities for YFP-GKC (n = 25) and YFP-GKC[trunc] (n = 26) molecules measured in transfected HEK 293 cells. (C) Population FR and EEFF measurements. (D and E) Fits of FR scatter plots for CFP-NSH3+YFP-GKC and CFP-NSH3+YFP-GKC[trunc] to a 1:1 binding model.
Mentions: We adopted the optical FRET two-hybrid approach (Erickson et al., 2003) to probe for interactions between split-domain CaVβ constructs (Fig. 4). This approach offered the advantage of reporting on the status of split-domain CaVβ SH3–GK interactions in the relevant setting of live HEK 293 cells, permitting a direct correlation of binding properties and functional electrophysiological experiments. Coding sequences for enhanced CFP and enhanced YFP were fused upstream of those for NSH3 and the two GK domain constructs, respectively. Confocal images of live cells show the subcellular localization of the distinct XFP-tagged proteins upon their cotransfection in HEK 293 cells (Fig. 4 A). CFP-NSH3 localized uniformly throughout the cell, with similar fluorescent intensity in the cytoplasm and the nucleus (Fig. 4 A, left). This distribution presumably reflects the fact that the relatively small size of CFP-NSH3 permits passive diffusion into the nucleus. By contrast, both YFP-GKC and YFP-GKC[trunc] were uniformly present in the cytosol, and excluded from the nucleus (Fig. 4 A, right). Importantly, the mean fluorescence intensities for YFP-GKC and YFP-GKC-[trunc] were not significantly different (Fig. 4 B; P = 0.07), indicating no dramatic differences in expression levels between these two proteins. This finding rules out the trivial possibility that the observed functional divergence between GKC and GKC[trunc] (Figs. 2 and 3) could be due to large differences in expression levels or protein stability between the two.

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
Related in: MedlinePlus