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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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NSH3 can interact independently with α1C. (A) Population FR and EEFF measurements for α1C-YFP+CFP-NSH3, α1C-YFP+CFP, and YFP-NSH3+CFP-α1C[I-II loop]. (B) Binding analyses on FR scatter plot for α1C-YFP+CFP-NSH3.
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fig7: NSH3 can interact independently with α1C. (A) Population FR and EEFF measurements for α1C-YFP+CFP-NSH3, α1C-YFP+CFP, and YFP-NSH3+CFP-α1C[I-II loop]. (B) Binding analyses on FR scatter plot for α1C-YFP+CFP-NSH3.

Mentions: The lack of effect of NSH3 on channel properties when cotransfected with GKC[ΔPYDVV] gave reason to wonder whether NSH3 could actually bind to α1C independently of an interaction with a CaVβ GK domain. Previous in vitro studies have indicated that the CaVβ SH3 domain may interact weakly with the α1C I-II loop (Maltez et al., 2005) or the carboxy tail (Gerhardstein et al., 2000), but an association with α1 subunit in the context of living cells has not been demonstrated. To address this, we probed for an interaction between CFP-NSH3 and YFP-α1C by FRET. We used an α1C subunit that was truncated at the carboxy terminus (α1C[1671]) to maximize the chances of observing FRET. Previous studies had shown that this truncated α1 was still dependent on CaVβ for trafficking and gating modulation (Erickson et al., 2001). Compared with control cells expressing CFP+YFP-α1C, test group cells coexpressing CFP-NSH3 and YFP-α1C displayed a significantly elevated FRET (FR = 1.68 ± 0.11, n = 9 for test-group cells; FR = 1.01 ± 0.04, n = 9 for control cells; P < 0.001). Therefore, NSH3 is able to independently associate with α1C in live HEK 293 cells. Furthermore, fits of the FRET data to a 1:1 binding model yielded a Kd,EFF = 11,708, indicating that the NSH3/α1C interaction was of a significantly lower affinity than the NSH3/GKC interaction. Moreover, this value is dramatically higher than one previously estimated for full-length CaVβ2a binding to α1C (Kd,EFF = 43) (Erickson et al., 2001). This is in agreement with the idea that the GK domain is primarily responsible for high-affinity binding of CaVβ to α1 subunits, compared with the SH3 domain. To determine whether NSH3 interacted with α1C primarily via the cytoplasmic domain I-II loop (Maltez et al., 2005), we investigated an association between YFP-NSH3 and CFP-α1C[I-II loop] by FRET. This experiment yielded a low FR value (Fig. 7 A, FR = 1.13 ± 0.06) that was not significantly different from the control case of YFP + CFP-NSH3 (Fig. 4 C, FR = 1.10 ± 0.04, P = 0.67). Hence the low-affinity interaction between NSH3 and the α1-subunit I-II loop may be below the threshold detectable by the FRET assay. Together with the identified FRET interaction between YFP-α1C and CFP-NSH3, the results suggest that NSH3 primarily binds to α1C at a site that is different from the I-II loop.


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)

NSH3 can interact independently with α1C. (A) Population FR and EEFF measurements for α1C-YFP+CFP-NSH3, α1C-YFP+CFP, and YFP-NSH3+CFP-α1C[I-II loop]. (B) Binding analyses on FR scatter plot for α1C-YFP+CFP-NSH3.
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fig7: NSH3 can interact independently with α1C. (A) Population FR and EEFF measurements for α1C-YFP+CFP-NSH3, α1C-YFP+CFP, and YFP-NSH3+CFP-α1C[I-II loop]. (B) Binding analyses on FR scatter plot for α1C-YFP+CFP-NSH3.
Mentions: The lack of effect of NSH3 on channel properties when cotransfected with GKC[ΔPYDVV] gave reason to wonder whether NSH3 could actually bind to α1C independently of an interaction with a CaVβ GK domain. Previous in vitro studies have indicated that the CaVβ SH3 domain may interact weakly with the α1C I-II loop (Maltez et al., 2005) or the carboxy tail (Gerhardstein et al., 2000), but an association with α1 subunit in the context of living cells has not been demonstrated. To address this, we probed for an interaction between CFP-NSH3 and YFP-α1C by FRET. We used an α1C subunit that was truncated at the carboxy terminus (α1C[1671]) to maximize the chances of observing FRET. Previous studies had shown that this truncated α1 was still dependent on CaVβ for trafficking and gating modulation (Erickson et al., 2001). Compared with control cells expressing CFP+YFP-α1C, test group cells coexpressing CFP-NSH3 and YFP-α1C displayed a significantly elevated FRET (FR = 1.68 ± 0.11, n = 9 for test-group cells; FR = 1.01 ± 0.04, n = 9 for control cells; P < 0.001). Therefore, NSH3 is able to independently associate with α1C in live HEK 293 cells. Furthermore, fits of the FRET data to a 1:1 binding model yielded a Kd,EFF = 11,708, indicating that the NSH3/α1C interaction was of a significantly lower affinity than the NSH3/GKC interaction. Moreover, this value is dramatically higher than one previously estimated for full-length CaVβ2a binding to α1C (Kd,EFF = 43) (Erickson et al., 2001). This is in agreement with the idea that the GK domain is primarily responsible for high-affinity binding of CaVβ to α1 subunits, compared with the SH3 domain. To determine whether NSH3 interacted with α1C primarily via the cytoplasmic domain I-II loop (Maltez et al., 2005), we investigated an association between YFP-NSH3 and CFP-α1C[I-II loop] by FRET. This experiment yielded a low FR value (Fig. 7 A, FR = 1.13 ± 0.06) that was not significantly different from the control case of YFP + CFP-NSH3 (Fig. 4 C, FR = 1.10 ± 0.04, P = 0.67). Hence the low-affinity interaction between NSH3 and the α1-subunit I-II loop may be below the threshold detectable by the FRET assay. Together with the identified FRET interaction between YFP-α1C and CFP-NSH3, the results suggest that NSH3 primarily binds to α1C at a site that is different from the I-II loop.

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