Radhakrishnan Kayalvizhi, Kamp Marcel A, Siapich Siarhei A, Hescheler Jürgen, Lüke Matthias, Schneider Toni
Institute of Neurophysiology, Center of Molecular Medicine Cologne, University of Cologne, Robert-Koch-Strasse 39, Cologne, Germany.
Cell Physiol Biochem. 2011;27(5):421-32. doi: 10.1159/000329963. Epub 2011 Jun 15.
Calcium channels are essential in coupling action potential to signal transduction in cells. There are several types of calcium channels, which can be pharmacologically classified as L-, N-, P/Q-, R- and T-type. But molecular basis of R-type channels is less clearly understood compared the other channel types. Therefore the current study aims at understanding the molecular functions of R-type calcium channels by identifying interaction partners of the channel.
In order to do so, a yeast two hybrid (Y2H) screen, with carboxy terminus of α1 subunit of the channel, as the bait, was performed. G1 subunit of v-ATPase was identified as a putative interaction partner of human Ca(v)2.3 by using the Y2H screening. The interaction was confirmed by immunoprecipitation. To study the functional importance of the interaction, bafilomycin A(1), a potent and specific inhibitor of v-ATPase was used in patch-clamp recordings in Ca(v)2.3 stably-transfected HEK-293 cells (2C6) as well as in electroretinography of the isolated bovine retina expressing R-type Ca(2+) channels.
G1 subunit of v-ATPase interacts with C-terminal tail of Ca(v)2.3 and bafilomycin A(1) reduces Ca(v)2.3 mediated calcium currents. Additionally peak I(Ca) is inhibited in retinal signal transduction when recorded as ERG b-wave.
The results suggest that v-ATPase interacts physically and also functionally with Ca(v)2.3. This is the first demonstration of association of Ca(v)2.3 C-terminus with a protein complex which is involved in transmembrane signalling.
钙通道在细胞中将动作电位与信号转导偶联过程中至关重要。钙通道有几种类型，可从药理学上分为L型、N型、P/Q型、R型和T型。但与其他通道类型相比，R型通道的分子基础了解得还不够清楚。因此，本研究旨在通过鉴定该通道的相互作用伙伴来了解R型钙通道的分子功能。
为此，以该通道α1亚基的羧基末端为诱饵进行了酵母双杂交（Y2H）筛选。通过Y2H筛选，v-ATP酶的G1亚基被鉴定为人Ca(v)2.3的一个假定相互作用伙伴。通过免疫沉淀证实了这种相互作用。为了研究这种相互作用的功能重要性，在稳定转染Ca(v)2.3的HEK-293细胞（2C6）的膜片钳记录以及表达R型Ca(2+)通道的离体牛视网膜的视网膜电图中使用了巴弗洛霉素A(1)，一种v-ATP酶的强效特异性抑制剂。
v-ATP酶的G1亚基与Ca(v)2.3的C末端尾巴相互作用，并且巴弗洛霉素A(1)降低了Ca(v)2.3介导的钙电流。此外，当记录为视网膜电图b波时，视网膜信号转导中的峰值I(Ca)受到抑制。
结果表明v-ATP酶在物理和功能上均与Ca(v)2.3相互作用。这是首次证明Ca(v)2.3 C末端与参与跨膜信号传导的蛋白质复合物存在关联。
