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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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Ablation of the SH3–GK association abolishes synergistic functional interactions between CaVβ SH3 and GK domains. (A) Exemplar current trace from a recombinant L-type channel reconstituted with GKC[ΔPYDVV] alone was evoked at a test potential of +50 mV. Tail current was measured at a −50-mV repolarization potential. Exemplar current from a channel reconstituted with NSH3+GKC is reproduced (gray trace) to facilitate direct visual comparison. (B) Population J–V relationship for GKC[ΔPYDVV]-reconstituted channels. Data for NSH3+GKC is reproduced (gray trace) for comparison. (C) Normalized tail G–V relationship for GKC[ΔPYDVV]-reconstituted channels (▴). Double-Boltzmann fit to the data (smooth curve) had the following parameters: Flow = 0.75, V1/2,low = 10.9 mV, V1/2,high = 74.3 mV, klow = 13.4 mV, khigh = 11.0 mV. Corresponding fit for NSH3+GKC is reproduced (gray trace) for comparison. (D–F) Data for channels reconstituted with NSH3+GKC[ΔPYDVV]. Identical format as A–C. Double-Boltzmann fit in F had the following parameters: Flow = 0.82, V1/2,low = 7.4 mV, V1/2,high = 72.9 mV, klow = 15.9 mV, khigh = 9.6 mV.
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fig6: Ablation of the SH3–GK association abolishes synergistic functional interactions between CaVβ SH3 and GK domains. (A) Exemplar current trace from a recombinant L-type channel reconstituted with GKC[ΔPYDVV] alone was evoked at a test potential of +50 mV. Tail current was measured at a −50-mV repolarization potential. Exemplar current from a channel reconstituted with NSH3+GKC is reproduced (gray trace) to facilitate direct visual comparison. (B) Population J–V relationship for GKC[ΔPYDVV]-reconstituted channels. Data for NSH3+GKC is reproduced (gray trace) for comparison. (C) Normalized tail G–V relationship for GKC[ΔPYDVV]-reconstituted channels (▴). Double-Boltzmann fit to the data (smooth curve) had the following parameters: Flow = 0.75, V1/2,low = 10.9 mV, V1/2,high = 74.3 mV, klow = 13.4 mV, khigh = 11.0 mV. Corresponding fit for NSH3+GKC is reproduced (gray trace) for comparison. (D–F) Data for channels reconstituted with NSH3+GKC[ΔPYDVV]. Identical format as A–C. Double-Boltzmann fit in F had the following parameters: Flow = 0.82, V1/2,low = 7.4 mV, V1/2,high = 72.9 mV, klow = 15.9 mV, khigh = 9.6 mV.

Mentions: To determine the functional effects of ablating the CaVβ SH3–GK interaction we turned to whole-cell electrophysiological experiments. GKC[ΔPYDVV] expressed alone with α1C was deficient in its ability to rescue whole-cell currents (Fig. 6, A and B; Table I) and to induce a hyperpolarizing shift in activation gating (Fig. 6 C; Table I), despite its demonstrated normal binding to the α1C[I-II loop] (Fig. 5 E). These results confirm our previous finding that the GK domain is insufficient to reconstitute the bulk of CaVβ modulatory properties in HEK 293 cells (Takahashi et al., 2004). The new insight came with studying the effects of NSH3+GKC [ΔPYDVV] on Ca2+ channel currents. Channels reconstituted with NSH3+GKC[ΔPYDVV] behaved similarly to those obtained with GKC[ΔPYDVV] alone, with respect to whole-cell current amplitude (Fig. 6, D and E; Table I) and the voltage dependence of channel activation (Fig. 6 F; Table I). These results contrast sharply with the synergism observed between NSH3 and GKC (Takahashi et al., 2004), and revealed that the SH3–GK interaction is requisite for CaVβ modulation of channel trafficking and gating. Overall, these results are consistent with a co-dependent model for CaVβ SH3 and GK domains to modulate channel function.


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)

Ablation of the SH3–GK association abolishes synergistic functional interactions between CaVβ SH3 and GK domains. (A) Exemplar current trace from a recombinant L-type channel reconstituted with GKC[ΔPYDVV] alone was evoked at a test potential of +50 mV. Tail current was measured at a −50-mV repolarization potential. Exemplar current from a channel reconstituted with NSH3+GKC is reproduced (gray trace) to facilitate direct visual comparison. (B) Population J–V relationship for GKC[ΔPYDVV]-reconstituted channels. Data for NSH3+GKC is reproduced (gray trace) for comparison. (C) Normalized tail G–V relationship for GKC[ΔPYDVV]-reconstituted channels (▴). Double-Boltzmann fit to the data (smooth curve) had the following parameters: Flow = 0.75, V1/2,low = 10.9 mV, V1/2,high = 74.3 mV, klow = 13.4 mV, khigh = 11.0 mV. Corresponding fit for NSH3+GKC is reproduced (gray trace) for comparison. (D–F) Data for channels reconstituted with NSH3+GKC[ΔPYDVV]. Identical format as A–C. Double-Boltzmann fit in F had the following parameters: Flow = 0.82, V1/2,low = 7.4 mV, V1/2,high = 72.9 mV, klow = 15.9 mV, khigh = 9.6 mV.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2266626&req=5

fig6: Ablation of the SH3–GK association abolishes synergistic functional interactions between CaVβ SH3 and GK domains. (A) Exemplar current trace from a recombinant L-type channel reconstituted with GKC[ΔPYDVV] alone was evoked at a test potential of +50 mV. Tail current was measured at a −50-mV repolarization potential. Exemplar current from a channel reconstituted with NSH3+GKC is reproduced (gray trace) to facilitate direct visual comparison. (B) Population J–V relationship for GKC[ΔPYDVV]-reconstituted channels. Data for NSH3+GKC is reproduced (gray trace) for comparison. (C) Normalized tail G–V relationship for GKC[ΔPYDVV]-reconstituted channels (▴). Double-Boltzmann fit to the data (smooth curve) had the following parameters: Flow = 0.75, V1/2,low = 10.9 mV, V1/2,high = 74.3 mV, klow = 13.4 mV, khigh = 11.0 mV. Corresponding fit for NSH3+GKC is reproduced (gray trace) for comparison. (D–F) Data for channels reconstituted with NSH3+GKC[ΔPYDVV]. Identical format as A–C. Double-Boltzmann fit in F had the following parameters: Flow = 0.82, V1/2,low = 7.4 mV, V1/2,high = 72.9 mV, klow = 15.9 mV, khigh = 9.6 mV.
Mentions: To determine the functional effects of ablating the CaVβ SH3–GK interaction we turned to whole-cell electrophysiological experiments. GKC[ΔPYDVV] expressed alone with α1C was deficient in its ability to rescue whole-cell currents (Fig. 6, A and B; Table I) and to induce a hyperpolarizing shift in activation gating (Fig. 6 C; Table I), despite its demonstrated normal binding to the α1C[I-II loop] (Fig. 5 E). These results confirm our previous finding that the GK domain is insufficient to reconstitute the bulk of CaVβ modulatory properties in HEK 293 cells (Takahashi et al., 2004). The new insight came with studying the effects of NSH3+GKC [ΔPYDVV] on Ca2+ channel currents. Channels reconstituted with NSH3+GKC[ΔPYDVV] behaved similarly to those obtained with GKC[ΔPYDVV] alone, with respect to whole-cell current amplitude (Fig. 6, D and E; Table I) and the voltage dependence of channel activation (Fig. 6 F; Table I). These results contrast sharply with the synergism observed between NSH3 and GKC (Takahashi et al., 2004), and revealed that the SH3–GK interaction is requisite for CaVβ modulation of channel trafficking and gating. Overall, these results are consistent with a co-dependent model for CaVβ SH3 and GK domains to modulate channel function.

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