The Electrically Silent Kv6.4 Subunit Confers Hyperpolarized Gating Charge Movement in Kv2.1/Kv6.4 Heterotetrameric Channels

The voltage-gated K+ (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ∼40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K+ equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits.电压门控钾离子（voltage-gated K+，Kv）通道亚基Kv6.4无法形成具有功能的同源四聚体通道（homotetrameric channels），而是与Kv2.1共同组装为具有功能的Kv2.1/Kv6.4异源四聚体通道（heterotetrameric channels）。相较于Kv2.1同源四聚体，Kv6.4可使Kv2.1/Kv6.4异源四聚体通道的失活电压依赖性产生约40 mV的超极化偏移，且对激活门控过程无显著影响。然而，Kv6.4介导的Kv2.1通道失活调控的潜在机制，以及Kv6.4亚基是否参与异源四聚体通道的电压依赖性门控过程，目前尚未得到充分阐明。本研究通过对表达上述通道的哺乳动物细胞开展门控电流（gating current）记录，发现相较于Kv2.1同源四聚体，Kv2.1/Kv6.4异源四聚体通道具备独特的门控电荷移动（gating charge movement）特征。Kv2.1/Kv6.4异源四聚体通道的门控电荷移动在生理性钾离子平衡电位附近存在额外组分，其特征为门控电荷移动的电压依赖性呈现第二条S型曲线关系。这种独特的门控电荷位移反映了Kv6.4电压感受结构域（voltage-sensing domain）的移动，且其电压依赖性与Kv2.1/Kv6.4通道失活的超极化偏移相匹配。上述研究结果为沉默型Kv6.4亚基调控Kv2.1通道失活门控动力学提供了机制层面的依据。