The transmitter release-site CaV2.2 channel cluster is linked to an endocytosis coat protein complex

Rajesh Khanna, Qi Li, Lyanne C. Schlichter, Elise F. Stanley

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Synaptic vesicles (SVs) are triggered to fuse with the surface membrane at the presynaptic transmitter release site (TRSs) core by Ca2+ influx through nearby attached CaV2.2 channels [see accompanying paper: Khanna et al. (2007) Eur. J. Neurosci., 26, 547-559] and are then recovered by endocytosis. In this study we test the hypothesis that the TRS core is linked to an endocytosis-related protein complex. This was tested by immunostaining analysis of the chick ciliary ganglion calyx presynaptic terminal and biochemical analysis of synaptosome lysate, using CaV2.2 as a marker for the TRS. We noted that CaV2.2 clusters abut heavy-chain (H)-clathrin patches at the transmitter release face. Quantitative coimmunostaining analysis (ICA/ICQ method) demonstrated a strong covariance of release-face CaV2.2 staining with that for the AP180 and intersectin endocytosis adaptor proteins, and a moderate covariance with H- or light-chain (L)-clathrin and dynamin coat proteins, consistent with a multimolecular complex. This was supported by coprecipitation of these proteins with CaV2.2 from brain synaptosome lysate. Interestingly, the channel neither colocalized nor coprecipitated with the endocytosis cargo-capturing adaptor AP2, even though this protein both colocalized and coprecipitated with H-clathrin. Fractional recovery analysis of the immunoprecipitated CaV2.2 complex by exposure to high NaCl (∼1 m) indicated that AP180 and S-intersectin adaptors are tightly bound to CaV2.2 while L-intersectin, H- and L-clathrin and dynamin form a less tightly linked subcomplex. Our results are consistent with two distinct clathrin endocytosis complexes: an AP2-containing, remote, non-TRS complex and a specialised, AP2-lacking, TRS-associated subcomplex linked via a molecular bridge. The most probable role of this subcomplex is to facilitate SV recovery after transmitter release.

Original languageEnglish (US)
Pages (from-to)560-574
Number of pages15
JournalEuropean Journal of Neuroscience
Volume26
Issue number3
DOIs
StatePublished - Aug 2007
Externally publishedYes

Fingerprint

Capsid Proteins
Endocytosis
Clathrin
Dynamins
Synaptosomes
Synaptic Vesicles
Clathrin Light Chains
Proteins
Clathrin Heavy Chains
Parasympathetic Ganglia
Presynaptic Terminals
Staining and Labeling
Membranes
Brain
intersectin 1

Keywords

  • Active zone
  • Calcium channel
  • Endocytosis
  • Presynaptic
  • Release site
  • Scaffold

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

The transmitter release-site CaV2.2 channel cluster is linked to an endocytosis coat protein complex. / Khanna, Rajesh; Li, Qi; Schlichter, Lyanne C.; Stanley, Elise F.

In: European Journal of Neuroscience, Vol. 26, No. 3, 08.2007, p. 560-574.

Research output: Contribution to journalArticle

Khanna, Rajesh ; Li, Qi ; Schlichter, Lyanne C. ; Stanley, Elise F. / The transmitter release-site CaV2.2 channel cluster is linked to an endocytosis coat protein complex. In: European Journal of Neuroscience. 2007 ; Vol. 26, No. 3. pp. 560-574.
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AB - Synaptic vesicles (SVs) are triggered to fuse with the surface membrane at the presynaptic transmitter release site (TRSs) core by Ca2+ influx through nearby attached CaV2.2 channels [see accompanying paper: Khanna et al. (2007) Eur. J. Neurosci., 26, 547-559] and are then recovered by endocytosis. In this study we test the hypothesis that the TRS core is linked to an endocytosis-related protein complex. This was tested by immunostaining analysis of the chick ciliary ganglion calyx presynaptic terminal and biochemical analysis of synaptosome lysate, using CaV2.2 as a marker for the TRS. We noted that CaV2.2 clusters abut heavy-chain (H)-clathrin patches at the transmitter release face. Quantitative coimmunostaining analysis (ICA/ICQ method) demonstrated a strong covariance of release-face CaV2.2 staining with that for the AP180 and intersectin endocytosis adaptor proteins, and a moderate covariance with H- or light-chain (L)-clathrin and dynamin coat proteins, consistent with a multimolecular complex. This was supported by coprecipitation of these proteins with CaV2.2 from brain synaptosome lysate. Interestingly, the channel neither colocalized nor coprecipitated with the endocytosis cargo-capturing adaptor AP2, even though this protein both colocalized and coprecipitated with H-clathrin. Fractional recovery analysis of the immunoprecipitated CaV2.2 complex by exposure to high NaCl (∼1 m) indicated that AP180 and S-intersectin adaptors are tightly bound to CaV2.2 while L-intersectin, H- and L-clathrin and dynamin form a less tightly linked subcomplex. Our results are consistent with two distinct clathrin endocytosis complexes: an AP2-containing, remote, non-TRS complex and a specialised, AP2-lacking, TRS-associated subcomplex linked via a molecular bridge. The most probable role of this subcomplex is to facilitate SV recovery after transmitter release.

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