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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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Distinctive channel modulation by split-domain CaVβ fragments differing in the split site. (A, top) Exemplar whole-cell Ba2+ currents evoked by a 25-ms test pulse to the indicated potentials from a HEK 293 cell transfected with α1C/NSH3/GKC. Holding potential was −90 mV. (A, bottom) Exemplar current from the same cell evoked with a +50-mV test pulse shows good isolation of the ON gating current. Tail current was measured at a −50-mV repolarization potential. (B) Exemplar currents from a cell transfected with α1C/NSH3/GKC[trunc]. Same format as A. (C) Population peak current versus voltage (J–V) relationship for recombinant L-type channels reconstituted with either NSH3+GKC (□, n = 9 for each data point) or NSH3+GKC[trunc] (▪, n = 11 for each data point). (D) Normalized tail G–V relationships for channels reconstituted with NSH3+GKC (▵) or NSH3+GKC[trunc] (▴). Smooth curves through the data are double-Boltzmann fits with the following parameters: for NSH3+GKC (Flow = 0.72, V1/2,low = −2.8 mV, V1/2,high = 56.1 mV, klow = 10.5 mV, khigh = 15.1 mV), for NSH3+GKC[trunc] (Flow = 0.69, V1/2,low = 3.8 mV, V1/2,high = 64.6 mV, klow = 12.5 mV, khigh = 11.0 mV). (E, top) Exemplar gating currents isolated at the reversal potential. (E, bottom) QON measurements, n = 6–11 cells for each construct. (F, left) Scatter plot of tail current amplitude (Itail) versus QON, and regression line (slope = 16.3 pA/fC; R2 = 0.95) for channels reconstituted with NSH3+GKC (▵). (F, right) Itail–QON scatter plot and regression line (black trace, slope = 5.7 pA/fC; R2 = 0.79) for NSH3+GKC[trunc]-reconstituted channels. Regression line for NSH3+GKC channels is reproduced (gray) to facilitate direct visual comparison.
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fig2: Distinctive channel modulation by split-domain CaVβ fragments differing in the split site. (A, top) Exemplar whole-cell Ba2+ currents evoked by a 25-ms test pulse to the indicated potentials from a HEK 293 cell transfected with α1C/NSH3/GKC. Holding potential was −90 mV. (A, bottom) Exemplar current from the same cell evoked with a +50-mV test pulse shows good isolation of the ON gating current. Tail current was measured at a −50-mV repolarization potential. (B) Exemplar currents from a cell transfected with α1C/NSH3/GKC[trunc]. Same format as A. (C) Population peak current versus voltage (J–V) relationship for recombinant L-type channels reconstituted with either NSH3+GKC (□, n = 9 for each data point) or NSH3+GKC[trunc] (▪, n = 11 for each data point). (D) Normalized tail G–V relationships for channels reconstituted with NSH3+GKC (▵) or NSH3+GKC[trunc] (▴). Smooth curves through the data are double-Boltzmann fits with the following parameters: for NSH3+GKC (Flow = 0.72, V1/2,low = −2.8 mV, V1/2,high = 56.1 mV, klow = 10.5 mV, khigh = 15.1 mV), for NSH3+GKC[trunc] (Flow = 0.69, V1/2,low = 3.8 mV, V1/2,high = 64.6 mV, klow = 12.5 mV, khigh = 11.0 mV). (E, top) Exemplar gating currents isolated at the reversal potential. (E, bottom) QON measurements, n = 6–11 cells for each construct. (F, left) Scatter plot of tail current amplitude (Itail) versus QON, and regression line (slope = 16.3 pA/fC; R2 = 0.95) for channels reconstituted with NSH3+GKC (▵). (F, right) Itail–QON scatter plot and regression line (black trace, slope = 5.7 pA/fC; R2 = 0.79) for NSH3+GKC[trunc]-reconstituted channels. Regression line for NSH3+GKC channels is reproduced (gray) to facilitate direct visual comparison.

Mentions: CaVβs contain SH3 and GK motifs in two conserved domains (Fig. 1, A and B). Recently, using a split-domain approach, we found that CaVβ fragments containing either the SH3 (NSH3) or GK (GKC) domains (Fig. 1 C) could reconstitute wild-type CaVβ functions only when both fragments were coexpressed with recombinant α1C (Takahashi et al., 2004). Fig. 2 A shows robust whole-cell currents from channels reconstituted with α1C/NSH3/GKC that confirms the previous results. Moreover, we and others found that NSH3 and GKC (or similar split-domain proteins) can interact in trans (Opatowsky et al., 2003; McGee et al., 2004; Takahashi et al., 2004), raising the question of whether and how the CaVβ SH3–GK domain interaction is important for channel modulation (Takahashi et al., 2004). The most direct way to address this issue would be to selectively interfere with the CaVβ SH3–GK interaction while leaving the GK–AID interaction unchanged.


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)

Distinctive channel modulation by split-domain CaVβ fragments differing in the split site. (A, top) Exemplar whole-cell Ba2+ currents evoked by a 25-ms test pulse to the indicated potentials from a HEK 293 cell transfected with α1C/NSH3/GKC. Holding potential was −90 mV. (A, bottom) Exemplar current from the same cell evoked with a +50-mV test pulse shows good isolation of the ON gating current. Tail current was measured at a −50-mV repolarization potential. (B) Exemplar currents from a cell transfected with α1C/NSH3/GKC[trunc]. Same format as A. (C) Population peak current versus voltage (J–V) relationship for recombinant L-type channels reconstituted with either NSH3+GKC (□, n = 9 for each data point) or NSH3+GKC[trunc] (▪, n = 11 for each data point). (D) Normalized tail G–V relationships for channels reconstituted with NSH3+GKC (▵) or NSH3+GKC[trunc] (▴). Smooth curves through the data are double-Boltzmann fits with the following parameters: for NSH3+GKC (Flow = 0.72, V1/2,low = −2.8 mV, V1/2,high = 56.1 mV, klow = 10.5 mV, khigh = 15.1 mV), for NSH3+GKC[trunc] (Flow = 0.69, V1/2,low = 3.8 mV, V1/2,high = 64.6 mV, klow = 12.5 mV, khigh = 11.0 mV). (E, top) Exemplar gating currents isolated at the reversal potential. (E, bottom) QON measurements, n = 6–11 cells for each construct. (F, left) Scatter plot of tail current amplitude (Itail) versus QON, and regression line (slope = 16.3 pA/fC; R2 = 0.95) for channels reconstituted with NSH3+GKC (▵). (F, right) Itail–QON scatter plot and regression line (black trace, slope = 5.7 pA/fC; R2 = 0.79) for NSH3+GKC[trunc]-reconstituted channels. Regression line for NSH3+GKC channels is reproduced (gray) to facilitate direct visual comparison.
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Related In: Results  -  Collection

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fig2: Distinctive channel modulation by split-domain CaVβ fragments differing in the split site. (A, top) Exemplar whole-cell Ba2+ currents evoked by a 25-ms test pulse to the indicated potentials from a HEK 293 cell transfected with α1C/NSH3/GKC. Holding potential was −90 mV. (A, bottom) Exemplar current from the same cell evoked with a +50-mV test pulse shows good isolation of the ON gating current. Tail current was measured at a −50-mV repolarization potential. (B) Exemplar currents from a cell transfected with α1C/NSH3/GKC[trunc]. Same format as A. (C) Population peak current versus voltage (J–V) relationship for recombinant L-type channels reconstituted with either NSH3+GKC (□, n = 9 for each data point) or NSH3+GKC[trunc] (▪, n = 11 for each data point). (D) Normalized tail G–V relationships for channels reconstituted with NSH3+GKC (▵) or NSH3+GKC[trunc] (▴). Smooth curves through the data are double-Boltzmann fits with the following parameters: for NSH3+GKC (Flow = 0.72, V1/2,low = −2.8 mV, V1/2,high = 56.1 mV, klow = 10.5 mV, khigh = 15.1 mV), for NSH3+GKC[trunc] (Flow = 0.69, V1/2,low = 3.8 mV, V1/2,high = 64.6 mV, klow = 12.5 mV, khigh = 11.0 mV). (E, top) Exemplar gating currents isolated at the reversal potential. (E, bottom) QON measurements, n = 6–11 cells for each construct. (F, left) Scatter plot of tail current amplitude (Itail) versus QON, and regression line (slope = 16.3 pA/fC; R2 = 0.95) for channels reconstituted with NSH3+GKC (▵). (F, right) Itail–QON scatter plot and regression line (black trace, slope = 5.7 pA/fC; R2 = 0.79) for NSH3+GKC[trunc]-reconstituted channels. Regression line for NSH3+GKC channels is reproduced (gray) to facilitate direct visual comparison.
Mentions: CaVβs contain SH3 and GK motifs in two conserved domains (Fig. 1, A and B). Recently, using a split-domain approach, we found that CaVβ fragments containing either the SH3 (NSH3) or GK (GKC) domains (Fig. 1 C) could reconstitute wild-type CaVβ functions only when both fragments were coexpressed with recombinant α1C (Takahashi et al., 2004). Fig. 2 A shows robust whole-cell currents from channels reconstituted with α1C/NSH3/GKC that confirms the previous results. Moreover, we and others found that NSH3 and GKC (or similar split-domain proteins) can interact in trans (Opatowsky et al., 2003; McGee et al., 2004; Takahashi et al., 2004), raising the question of whether and how the CaVβ SH3–GK domain interaction is important for channel modulation (Takahashi et al., 2004). The most direct way to address this issue would be to selectively interfere with the CaVβ SH3–GK interaction while leaving the GK–AID interaction unchanged.

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