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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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The split site, and not the loss of 81 residues, is responsible for functional deficiencies of channels reconstituted with GKC[trunc]. (A–F) Exemplar currents and population plots for channels reconstituted with NSH3-V2+GKC and NSH3-V2+GKC[trunc], respectively. Format identical to Fig. 2 legend. Smooth fits in D have the following parameters: for NSH3-V2+GKC (Flow = 0.70, V1/2,low = −2.6 mV, V1/2,high = 56.0 mV, klow = 10.6 mV, khigh = 14.5 mV), for NSH3-V2+GKC[trunc] (Flow = 0.75, V1/2,low = 16.1 mV, V1/2,high = 70.2 mV, klow = 17.0 mV, khigh = 9.6 mV). For regression lines in F, slope = 19.6 pA/fC, R2 = 0.98 for NSH3-V2+GKC; slope = 5.96 pA/fC, R2 = 0.89 for NSH3-V2+GKC[trunc] channels.
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fig3: The split site, and not the loss of 81 residues, is responsible for functional deficiencies of channels reconstituted with GKC[trunc]. (A–F) Exemplar currents and population plots for channels reconstituted with NSH3-V2+GKC and NSH3-V2+GKC[trunc], respectively. Format identical to Fig. 2 legend. Smooth fits in D have the following parameters: for NSH3-V2+GKC (Flow = 0.70, V1/2,low = −2.6 mV, V1/2,high = 56.0 mV, klow = 10.6 mV, khigh = 14.5 mV), for NSH3-V2+GKC[trunc] (Flow = 0.75, V1/2,low = 16.1 mV, V1/2,high = 70.2 mV, klow = 17.0 mV, khigh = 9.6 mV). For regression lines in F, slope = 19.6 pA/fC, R2 = 0.98 for NSH3-V2+GKC; slope = 5.96 pA/fC, R2 = 0.89 for NSH3-V2+GKC[trunc] channels.

Mentions: Functionally, electrophysiological data obtained from channels reconstituted with NSH3-V2+GKC[trunc] (Fig. 3) were essentially identical to that obtained with NSH3+GKC[trunc] (Table I). NSH3-V2+GKC[trunc] was unable to recover the bulk of whole-cell current amplitude (Fig. 3, A–C), did not recapitulate the hyperpolarizing shift in channel activation gating (Fig. 3 D), and did not restore the increased-Po property (Fig. 3 F, ▴; slope = 5.96 ± 1.69 pA/fC; P < 0.001 compared with NSH3-V2+GKC, ▵). However, NSH3-V2+GKC[trunc] fully restored the channel trafficking function (Fig. 3 E). By contrast, NSH3-V2+GKC recovered wild-type whole-cell current amplitude (Fig. 3, A–C) by reconstituting both the trafficking (Fig. 3 E) and enhanced-Po functions (Fig. 3 F, ▵), and also recapitulated the hyperpolarizing shift in the voltage dependence of channel activation (Fig. 3 D; Table I). Together, these results affirmed the unexpected result that a simple difference in the cut site of split-domain CaVβ constructs can uncouple the trafficking and gating-modulation functions. Moreover, these results ruled out the explanation that NSH3+GKC[trunc] did not recover the gating-modulation functions simply because of the mere absence of an 81–amino acid central segment of CaVβ2a. This raised the idea that functional disparities between channels reconstituted with NSH3 (or NSH3-V2) and GKC or GKC[trunc], respectively, might be due to differences in the status of the CaVβ SH3–GK domain interaction. Accordingly, we next sought direct evidence for potential variations in the way in which GKC and GKC[trunc] interacted with NSH3.


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)

The split site, and not the loss of 81 residues, is responsible for functional deficiencies of channels reconstituted with GKC[trunc]. (A–F) Exemplar currents and population plots for channels reconstituted with NSH3-V2+GKC and NSH3-V2+GKC[trunc], respectively. Format identical to Fig. 2 legend. Smooth fits in D have the following parameters: for NSH3-V2+GKC (Flow = 0.70, V1/2,low = −2.6 mV, V1/2,high = 56.0 mV, klow = 10.6 mV, khigh = 14.5 mV), for NSH3-V2+GKC[trunc] (Flow = 0.75, V1/2,low = 16.1 mV, V1/2,high = 70.2 mV, klow = 17.0 mV, khigh = 9.6 mV). For regression lines in F, slope = 19.6 pA/fC, R2 = 0.98 for NSH3-V2+GKC; slope = 5.96 pA/fC, R2 = 0.89 for NSH3-V2+GKC[trunc] channels.
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fig3: The split site, and not the loss of 81 residues, is responsible for functional deficiencies of channels reconstituted with GKC[trunc]. (A–F) Exemplar currents and population plots for channels reconstituted with NSH3-V2+GKC and NSH3-V2+GKC[trunc], respectively. Format identical to Fig. 2 legend. Smooth fits in D have the following parameters: for NSH3-V2+GKC (Flow = 0.70, V1/2,low = −2.6 mV, V1/2,high = 56.0 mV, klow = 10.6 mV, khigh = 14.5 mV), for NSH3-V2+GKC[trunc] (Flow = 0.75, V1/2,low = 16.1 mV, V1/2,high = 70.2 mV, klow = 17.0 mV, khigh = 9.6 mV). For regression lines in F, slope = 19.6 pA/fC, R2 = 0.98 for NSH3-V2+GKC; slope = 5.96 pA/fC, R2 = 0.89 for NSH3-V2+GKC[trunc] channels.
Mentions: Functionally, electrophysiological data obtained from channels reconstituted with NSH3-V2+GKC[trunc] (Fig. 3) were essentially identical to that obtained with NSH3+GKC[trunc] (Table I). NSH3-V2+GKC[trunc] was unable to recover the bulk of whole-cell current amplitude (Fig. 3, A–C), did not recapitulate the hyperpolarizing shift in channel activation gating (Fig. 3 D), and did not restore the increased-Po property (Fig. 3 F, ▴; slope = 5.96 ± 1.69 pA/fC; P < 0.001 compared with NSH3-V2+GKC, ▵). However, NSH3-V2+GKC[trunc] fully restored the channel trafficking function (Fig. 3 E). By contrast, NSH3-V2+GKC recovered wild-type whole-cell current amplitude (Fig. 3, A–C) by reconstituting both the trafficking (Fig. 3 E) and enhanced-Po functions (Fig. 3 F, ▵), and also recapitulated the hyperpolarizing shift in the voltage dependence of channel activation (Fig. 3 D; Table I). Together, these results affirmed the unexpected result that a simple difference in the cut site of split-domain CaVβ constructs can uncouple the trafficking and gating-modulation functions. Moreover, these results ruled out the explanation that NSH3+GKC[trunc] did not recover the gating-modulation functions simply because of the mere absence of an 81–amino acid central segment of CaVβ2a. This raised the idea that functional disparities between channels reconstituted with NSH3 (or NSH3-V2) and GKC or GKC[trunc], respectively, might be due to differences in the status of the CaVβ SH3–GK domain interaction. Accordingly, we next sought direct evidence for potential variations in the way in which GKC and GKC[trunc] interacted with NSH3.

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