Chen Huanmian, Puhl Henry L, Niu Shui-Lin, Mitchell Drake C, Ikeda Stephen R
Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Neurosci. 2005 Oct 19;25(42):9762-72. doi: 10.1523/JNEUROSCI.3111-05.2005.
Rad, Gem/Kir, Rem, and Rem2 are members of the Ras-related RGK (Rad, Gem, and Kir) family of small GTP-binding proteins. Heterologous expression of RGK proteins interferes with de novo calcium channel assembly/trafficking and dramatically decreases the amplitude of currents arising from preexisting high-voltage-activated calcium channels. These effects probably result from the direct interaction of RGK proteins with calcium channel beta subunits. Among the RGK family, Rem2 is the only member abundantly expressed in neuronal tissues. Here, we examined the ability of Rem2 to modulate endogenous voltage-activated calcium channels in rat sympathetic and dorsal root ganglion neurons. Heterologous expression of Rem2 nearly abolished calcium currents arising from preexisting high-voltage-activated calcium channels without affecting low-voltage-activated calcium channels. Rem2 inhibition of N-type calcium channels required both the Ras homology (core) domain and the polybasic C terminus. Mutation of a putative GTP/Mg2+ binding motif in Rem2 did not affect suppression of calcium currents. Loading neurons with GDP-beta-S via the patch pipette did not reverse Rem2-mediated calcium channel inhibition. Finally, [(125)I]Tyr22-omega-conotoxin GVIA cell surface binding in tsA201 cells stably expressing N-type calcium channels was not altered by Rem2 expression at a time when calcium current was totally abolished. Together, our results support a model in which Rem2 localizes to the plasma membrane via a C-terminal polybasic motif and interacts with calcium channel beta subunits in the preassembled N-type channel, thereby forming a nonconducting species.
Rad、Gem/Kir、Rem和Rem2是小GTP结合蛋白的Ras相关RGK（Rad、Gem和Kir）家族成员。RGK蛋白的异源表达会干扰新生钙通道的组装/运输，并显著降低由预先存在的高压激活钙通道产生的电流幅度。这些效应可能是由于RGK蛋白与钙通道β亚基的直接相互作用所致。在RGK家族中，Rem2是唯一在神经组织中大量表达的成员。在此，我们研究了Rem2调节大鼠交感神经和背根神经节神经元内源性电压激活钙通道的能力。Rem2的异源表达几乎消除了由预先存在的高压激活钙通道产生的钙电流，而不影响低压激活钙通道。Rem2对N型钙通道的抑制作用既需要Ras同源（核心）结构域，也需要多碱性C末端。Rem2中一个假定的GTP/Mg2+结合基序的突变并不影响对钙电流的抑制。通过膜片钳微量移液器向神经元加载GDP-β-S并不能逆转Rem2介导的钙通道抑制作用。最后，在钙电流完全消失时，Rem2的表达并未改变稳定表达N型钙通道的tsA201细胞中[(125)I]酪氨酸22-ω-芋螺毒素GVIA的细胞表面结合。总之，我们的结果支持一种模型，即Rem2通过C末端多碱性基序定位于质膜，并与预组装的N型通道中的钙通道β亚基相互作用，从而形成一种非传导性物质。
