Fluorescence measurements reveal stoichiometry of K+ channels formed by modulatory and delayed rectifier α-subunits
Fluorescence measurements reveal stoichiometry of K+ channels formed by modulatory and delayed rectifier α-subunits
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DOI：
10.1073/pnas.0500468102
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发表时间：
2005-04-26
影响因子：
11.1
通讯作者：
Stocker, M
中科院分区：
文献类型：
--
作者：
Kerschensteiner, D;Soto, F;Stocker, M
调节性 α 亚基占所有电压门控 K(+) 通道 (Kv) α 亚基的四分之一，不会组装成同聚通道，而是选择性地与延迟整流器 Kv2 亚基结合，形成化学计量未知的异聚通道。它们独特的表达模式和独特的功能特性使这些通道成为广泛的天然 K(+) 电流的候选分子相关物。在这里，我们结合 FRET 和电生理学测量来确定由延迟整流 Kv2.1 亚基和调节 Kv9.3 α 亚基组成的异聚通道的化学计量和几何形状。 Kv通道α亚基与GFP变体融合，并且研究了标记和未标记α亚基的不同组合的异聚化。通过受体光漂白评估的 FRET 仅在功能通道形成时观察到。我们的结果是从两个独立的实验范例中获得的，表明固定化学计量的异聚 Kv2.1/Kv9.3 通道的形成，由三个 Kv2.1 亚基和一个 Kv9.3 亚基组成。引人注目的是，尽管化学计量不均匀，我们发现异聚 Kv2.1/Kv9.3 通道在中心孔周围保持亚基的假对称排列。
Modulatory alpha-subunits, which comprise one-fourth of all voltage-gated K(+) channel (Kv) alpha-subunits, do not assemble into homomeric channels, but selectively associate with delayed rectifier Kv2 subunits to form heteromeric channels of unknown stoichiometry. Their distinct expression patterns and unique functional properties have made these channels candidate molecular correlates for a broad set of native K(+) currents. Here, we combine FRET and electrophysiological measurements to determine the stoichiometry and geometry of heteromeric channels composed of the delayed rectifier Kv2.1 subunit and the modulatory Kv9.3 alpha-subunit. Kv channel alpha-subunits were fused with GFP variants, and heteromerization of different combinations of tagged and untagged alpha-subunits was studied. FRET, evaluated by acceptor photobleaching, was only observed upon formation of functional channels. Our results, obtained from two independent experimental paradigms, suggest the formation of heteromeric Kv2.1/Kv9.3 channels of fixed stoichiometry consisting of three Kv2.1 subunits and one Kv9.3 subunit. Strikingly, despite this uneven stoichiometry, we find that heteromeric Kv2.1/Kv9.3 channels maintain a pseudosymmetric arrangement of subunits around the central pore.