Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado; Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado.
Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado; Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado; Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado.
Biophys J. 2022 Mar 1;121(5):755-768. doi: 10.1016/j.bpj.2022.01.021. Epub 2022 Jan 31.
Ion channels are well known for their ability to regulate the cell membrane potential. However, many ion channels also have functions that do not involve ion conductance. Kv2 channels are one family of ion channels whose non-conducting functions are central to mammalian cell physiology. Kv2.1 and Kv2.2 channels form stable contact sites between the endoplasmic reticulum and plasma membrane via an interaction with endoplasmic reticulum resident proteins. To perform this structural role, Kv2 channels are expressed at extremely high densities on the plasma membranes of many cell types, including central pyramidal neurons, α-motoneurons, and smooth muscle cells. Research from our lab and others has shown that the majority of these plasma membrane Kv2.1 channels do not conduct potassium in response to depolarization. The mechanism of this channel silencing is unknown but is thought to be dependent on channel density in the membrane. Furthermore, the prevalence of a non-conducting population of Kv2.2 channels has not been directly tested. In this work we make improved measurements of the numbers of conducting and non-conducting Kv2.1 channels expressed in HEK293 cells and expand the investigation of non-conducting channels to three additional Kv α-subunits: Kv2.2, Kv1.4, and Kv1.5. By comparing the numbers of gating and conducting channels in individual HEK293 cells, we found that on average, only 50% of both Kv2.1 and Kv2.2 channels conducted potassium and, as previously suggested, that fraction decreased with increased channel density in the plasma membrane. At the highest spatial densities tested, which are comparable with those found at Kv2 clusters in situ, only 20% of Kv2.1 and Kv2.2 channels conducted potassium. We also show for the first time that Kv1.4 and Kv1.5 exhibit density-dependent silencing, suggesting that this phenomenon has an underlying mechanism that is shared by Kv channels from multiple families.
离子通道以其调节细胞膜电位的能力而闻名。然而，许多离子通道也具有不涉及离子电导的功能。Kv2 通道是一类离子通道，其非传导功能是哺乳动物细胞生理学的核心。Kv2.1 和 Kv2.2 通道通过与内质网驻留蛋白相互作用，在内质网和质膜之间形成稳定的接触位点。为了发挥这种结构作用，Kv2 通道在许多细胞类型的质膜上以极高的密度表达，包括中枢锥体细胞、α运动神经元和平滑肌细胞。我们实验室和其他实验室的研究表明，这些质膜 Kv2.1 通道中的大多数在去极化时不传导钾。这种通道沉默的机制尚不清楚，但据信依赖于膜中的通道密度。此外，尚未直接测试非传导性 Kv2.2 通道的流行程度。在这项工作中，我们改进了对 HEK293 细胞中表达的可传导和非传导 Kv2.1 通道数量的测量，并将非传导通道的研究扩展到另外三个 Kv α-亚基：Kv2.2、Kv1.4 和 Kv1.5。通过比较单个 HEK293 细胞中门控和传导通道的数量，我们发现 Kv2.1 和 Kv2.2 通道的平均传导钾的比例仅为 50%，并且与质膜中通道密度增加的情况一样，该比例降低。在所测试的最高空间密度下，与原位 Kv2 簇中发现的密度相当，只有 20%的 Kv2.1 和 Kv2.2 通道传导钾。我们还首次表明 Kv1.4 和 Kv1.5 表现出密度依赖性沉默，表明这种现象具有由多个家族的 Kv 通道共享的潜在机制。
