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A dynamic -ß inter-subunit agonist signaling complex is a novel feedback mechanism for regulating L-type Ca²⁺ channel opening

Rong Zhang^*, Igor Dzhura^*, Chad E. Grueter, William Thiel, Roger J. Colbran and Mark E. Anderson^*,,1

^* Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA;
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA; and
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, USA

¹ Correspondence: M.E.A., E-mail: mark.anderson{at}vanderbilt.edu or R.J.C., E-mail: roger.colbran{at}vanderbilt.edu

SPECIFIC AIMS

Calmodulin binding peptides modeled after the L-type Ca²⁺ channel (Ca_V1.2) pore-forming subunit cytoplasmic C terminus increase channel openings when applied to the cytoplasmic face of native L-type Ca²⁺ channels voltage-clamped in excised cell membrane patches, suggesting these sequences are endogenous agonist ligands. The aim of this study was to test the hypothesis that the cytoplasmic ß subunit, which itself increases Ca_V1.2 channel opening probability and is constitutively bound to the subunit, was the receptor for these endogenous ligands.

PRINCIPAL FINDINGS

1. The subunit C terminus binds to the ß subunit through embedded calmodulin interacting motifs
We coexpressed various flag-tagged Ca_V1.2 subunit C terminus constructs (Fig. 1 a) with full-length myc-tagged ß subunits (ß_2a) in tsA-201 cells. Cell lysates were assayed for protein expression by immunoblotting (Fig. 1b , input) and run over anti-flag agarose to immunoprecipitate (IP) the subunit C terminus-derived proteins. IP subunit C terminus proteins were immunoblotted with anti-myc antibodies to probe for binding interactions (Fig. 1b ). The full-length subunit C terminus bound ß (Fig. 1b , IP lane 2), as did the CaM binding CB/IQ domain alone (Fig. 1b , IP lane 3). In contrast, the subunit C terminus lacking the CB/IQ domain failed to bind the ß subunit (Fig. 1b , IP lane 4). To test whether this novel CB/IQ to ß subunit binding disrupted the constitutive binding between the subunit (via the interacting domain, AID in the cytoplasmic I-II linker domain) and the ß subunit, we coexpressed HA-tagged proteins with and without AID (Fig. 1c ) with myc-tagged ß subunits and flag-tagged CB/IQ peptides. Cell lysates were IP with anti-flag agarose and probed with anti-myc and anti-HA antibodies to reveal simultaneous CB/IQ to ß and AID to ß subunit binding (Fig. 1d , lane 4). Further studies mapped the critical residues for CB/IQ binding to the ß subunit to the Src homology 3 (SH3) -containing ß subunit N terminus (residues 1-212), while the ß subunit residues 213-604, containing a guanylate kinase-like domain (GK) were sufficient for binding the subunit AID (Fig. 3, full manuscript).

View larger version (35K): [in this window] [in a new window]   Figure 1. Bivalent interactions of the LTCC ß subunit with the subunit I-II cytoplasmic linker and C terminus CaM binding domains. a) Schematic depictions of the subunit with FLAG epitope-labeled C terminus-derived constructs used for pull down assays (b). b) Lysates of tsA-201 cells containing myc-tagged ß subunit and FLAG-tagged subunit C terminus constructs were immunoblotted to detect expression levels (top panels marked as input). Labeled red arrows indicate the mobility and lane location of each expressed C terminus protein. Black arrowhead at the left shows a nonspecific binding pattern that was consistently present with the anti-FLAG antibody (Ab). The lowermost panel shows the ß subunit coimmunoprecipitated (IP) by immobilized FLAG epitope-tagged proteins with the whole C terminus (lane 2) and the CB/IQ bounded region of the C terminus (lane 3) but not with FLAG vector alone (lane 1) or with the C terminus lacking CB/IQ (lane 4). Molecular mass (kDa) is indicated to the right. c) Schematic drawings of subunit I-II cytoplasmic linker domain constructs. d) tsA-201 cell extracts containing FLAG-tagged CB/IQ domain, myc-tagged ß subunit, and/or the indicated HA-tagged I-II linker domain construct (c) were immunoblotted to detect expression levels (top panels labeled input). FLAG-tagged CB/IQ domains were immunoprecipitated (IP) from lysates using anti-FLAG agarose and immunoblotted with antibodies to the myc and HA tag (lower panels). The ß subunit coimmunoprecipitated with CB/IQ (lanes 3–5), but ß coimmunoprecipitated only with the I-II linker domain containing the AID (lane 4). Labeled red arrows indicate the mobility and lane location of the myc-tagged ß subunit and the HA-tagged I-II linker protein. Molecular weight markers are on the right; arrowheads indicate IgG heavy and light chains.

2. Calmodulin overexpression prevents CB/IQ motifs from binding to the ß subunit and prevents CB/IQ agonist actions at L-type Ca²⁺ channels in excised cardiomyocyte cell membranes
We performed coexpression studies with flag-tagged calmodulin binding CB/IQ motifs and myc-tagged ß subunits, in the absence and presence of calmodulin overexpression. Calmodulin overexpression significantly reduced CB/IQ binding to full-length ß and to the ß N terminus residues 1-212 (Fig. 4, full manuscript). To test whether the negative regulation of CB/IQ to ß binding by calmodulin was functionally significant, we measured single channel opening probability in voltage-clamped cell membrane patches excised from cardiomyocytes, where and ß subunits are both present. CB/IQ peptide (15–18 µM) significantly increased channel opening probability (8.3±1.9%, P=0.037) compared with control (3.0±0.8%), but this agonist action of CB/IQ was blocked by the addition of calmodulin (15–18 µM, 3.4±0.6%) (Fig. 1 , full manuscript).

3. The ß subunit is required for increased L-type Ca²⁺ channel openings in response to CB/IQ motifs and this agonist action is negatively regulated by calmodulin
We measured the effects of CB/IQ on single channel opening in excised cell membrane patches from tsA-201 cells, with and ß subunit coexpression or subunit expression alone. CB/IQ agonist responses were present with L-type Ca²⁺ channels consisting of and ß subunits (Fig. 2 a, b) but absent when channels lacked the ß subunit (Fig. 2c ). Increases in opening probability in channels with and ß subunits by CB/IQ were prevented by addition of calmodulin (Fig. 2a, b ), similar to observations in native L-type Ca²⁺ channels (Fig. 1 , full manuscript). L-type Ca²⁺ channels composed of and ß subunits or subunits alone were both functional, as seen by equivalent responses to the agonist drug BayK-8644, which acts through a binding site on the subunit (Fig. 2d ). CaM binding peptides derived from CaMKII (290-309) did not increase Ca²⁺ channel openings or affect CB/IQ agonist actions, indicating that CB/IQ effects on Ca²⁺ channel activity were specific and distinct from the CaM binding properties (Fig. 2e ). On the other hand, coexpression of with ß subunits lacking the CB/IQ binding sequence (residues 1-212) resulted in Ca²⁺ channels with reduced basal opening probability that were unresponsive to CB/IQ (Fig. 2f ).

View larger version (36K): [in this window] [in a new window]   Figure 2. CB/IQ increases LTCC opening through a ß subunit-dependent mechanism that is negatively regulated by CaM. a) Raw data tracings showing unitary LTCC currents recorded from excised tsA-201 cell membrane patches with and ß subunit coexpression. The indicated GST proteins were added at 15–18 µM without or with a 1:1 molar ratio of CaM. Addition of GST-CB/IQ caused a marked increase in channel opening compared with control (GST alone) and addition of CaM blocked this effect. Summary data for LTCC opening probability (Po) recorded from cell membrane patches excised from tsA-201 cells coexpressing and ß subunits (b) or expressing subunit alone (c) (P=0.003 vs. all other groups). d) The LTCC agonist agent BayK 8644 markedly increased Po in the presence (black bar) or absence (open bar) of ß subunit coexpression. e) A CaM binding peptide (290-309) did not affect basal activity in LTCCs reconstituted from and ß subunit coexpression compared with control, nor did it reduce the agonist effect of CB/IQ. However, LTCC Po was significantly greater (P<0.001) in CB/IQ or 290-309+CB/IQ-treated membrane patches than in control or 290-309-treated membrane patches. The data from control and CB/IQ-treated membrane patches are the same as shown in panel b). f) Basal LTCC activity was reduced by coexpression of a ß subunit lacking the CB/IQ binding region but still competent to bind the subunit I-II linker; activity of these LTCCs did not increase with CB/IQ.

CONCLUSIONS AND SIGNIFICANCE

L-type Ca²⁺ channels are macromolecular protein complexes in neurons and myocytes that open in response to cell membrane depolarization to supply Ca²⁺ for regulating gene transcription, vesicle secretion, and triggering cell contraction. L-type Ca²⁺ channels include a pore-forming and an auxiliary ß subunit; subunit openings are regulated by cellular Ca²⁺ through a mechanism involving the Ca²⁺-sensing protein calmodulin and calmodulin binding CB/IQ motifs in the subunit cytoplasmic C terminus. We have shown that calmodulin binding motifs embedded within the highly mobile subunit cytoplasmic C terminus are also endogenous agonist ligands that increase L-type Ca²⁺ channel openings under low calmodulin activity conditions by binding the ß subunit. This novel -ß subunit interaction is mechanistically distinct from constitutive to ß binding, which is mediated by AID, because CB/IQ binding does not disrupt AID to ß binding (Fig. 1d ). On the other hand, CB/IQ to ß subunit binding is inhibited by calmodulin and CB/IQ agonist actions are dynamic and negatively regulated by calmodulin (Fig. 2) . This bifunctional nature of CB/IQ supports a new model of feedback regulation at L-type Ca²⁺ channels (Fig. 3 ) where endogenous agonist domains increase Ca²⁺ entry. This mechanism for increasing Ca²⁺ entry is subject to negative feedback by increased activity of Ca²⁺-bound calmodulin that occludes the CB/IQ agonist signal to reduce Ca²⁺ channel opening.

View larger version (29K): [in this window] [in a new window]   Figure 3. The experimental approach for single channel recording and a model of the proposed mechanism for feedback regulation of L-type Ca2+ channels by the CB/IQ region in the subunit C terminus. a) A schematic depiction of the experimental paradigm for electrophysiological studies. Unitary LTCC currents recorded from excised cell membrane patches are measured in Ba2+-containing solution because the current from individual channel openings is too small to resolve with Ca2+ as charge carrier. The bulk bath (cytoplasmic) Ca2+ is "clamped" at 200 nM, while access to the bath allows controlled addition of macromolecules to the cytoplasmic LTCC face. b) A schematic depiction of the LTCC and ß subunits. The interacting domain (AID) resides on the cytoplasmic region linking the homologous membrane-bound regions I and II and is required for constitutive -ß subunit binding. The cytosolic subunit C-terminal domain contains three calmodulin (CaM) binding domains, including the CB (denotes calmodulin binding) and IQ (for characteristic Ile and Gln residues) domains studied here. c) The CB/IQ peptide (red cylinder) binds to the ß subunit and increases LTTC opening probability under low Ca2+/CaM conditions (left panel) but high Ca2+/CaM binds the CB/IQ peptide and inhibits ß subunit binding and agonist signaling (right panel). The fact that the CB/IQ peptide enhances LTCC openings under low Ca2+/CaM conditions indicates that the peptide is a superior agonist compared with the endogenous CB/IQ motif, perhaps because it is not constrained by structural elements present within the subunit C terminus. d) The hypothesized mechanism for CB/IQ feedback regulation of LTCC opening is shown. CaM is constitutively tethered to the subunit C terminus but at low Ca2+ does not bind to the CB/IQ motif allowing for ß subunit binding and increased LTCC opening (left panel). When Ca2+ is increased Ca2+/CaM binds to and occludes the CB/IQ motif to prevent ß subunit binding and reduced LTCC opening (right panel).

It is now well established that calmodulin can associate with a wide variety of voltage-gated ion channel proteins, including L-type Ca²⁺ channels, to confer Ca²⁺ responsiveness to channel opening. The CB/IQ motifs have been identified as regulatory sites for increasing Ca²⁺ channel openings but the mechanism of action has been uncertain. Our data provide a mechanistic framework for understanding CB/IQ signaling in L-type Ca²⁺ channels and suggest the possibility that a similar mechanism is operative in other voltage-gated ion channels with C terminus calmodulin binding sites.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3283fje;

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