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J. Biol. Chem., Vol. 278, Issue 49, 49386-49400, December 5, 2003

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Competitive and Synergistic Interactions of G Protein ₂ and Ca²⁺ Channel _1b Subunits with Ca_v2.1 Channels, Revealed by Mammalian Two-hybrid and Fluorescence Resonance Energy Transfer Measurements^*

Alexander Hümmer, Oliver Delzeith, Shannon R. Gomez, Rosa L. Moreno, Melanie D. Mark, and Stefan Herlitze

From the Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4975

Presynaptic Ca²⁺ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein subunits modulate the binding of the Ca²⁺ channel subunit on the Ca²⁺ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca²⁺ channel and G protein subunits on the two separate binding sites, i.e. the loopI–II and the C terminus, and on the full-length P/Q-type ₁2.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca²⁺ channel _1b subunit induces a strong fluorescent signal when interacting with the loopI–II but not with the C terminus. In contrast, the G protein subunit induces FRET signals on both the C terminus and loopI–II. Analysis of the interactions on the full-length channel indicates that Ca²⁺ channel _1b and G protein subunits bind to the ₁ subunit at the same time. Coexpression of the G protein increases the FRET signal between ₁/_1b FRET pairs but not for ₁/_1b FRET pairs where the C terminus was deleted from the ₁ subunit. The results suggest that the G protein alters the orientation and/or association between the Ca²⁺ channel and ₁2.1 subunits, which involves the C terminus of the ₁ subunit and may corresponds to a new conformational state of the channel.

Received for publication, June 23, 2003 , and in revised form, September 22, 2003.

^* This work was supported by National Institutes of Health Grant R01 NS42623-01A1 (to S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains 2 tables.

To whom correspondence should be addressed: Dept. of Neurosciences, Case Western Reserve University, School of Medicine, Rm. E604, 10900 Euclid Ave., Cleveland, OH 44106-4975. Tel.: 216-368-1804; Fax: 216-368-4650; E-mail: sxh106{at}pop.cwru.edu.

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