The electrically silent Kv5.1 subunit forms heteromeric Kv2 channels in cortical neurons and confers distinct functional properties.

Abstract

Voltage-gated K⁺ channels of the Kv2 family are highly expressed in brain and play dual roles in regulating neuronal excitability and in organizing endoplasmic reticulum - plasma membrane (ER-PM) junctions. Studies in heterologous cells suggest that Kv2.1 and Kv2.2 co-assemble with "electrically silent" KvS subunits to form heterotetrameric channels with distinct biophysical properties, but the prevalence and localization of these channels in native neurons is unknown. Here, using mass spectrometry-based proteomics, we identified five KvS subunits as components of native Kv2.1 channels immunopurified from mouse brain of both sexes, the most abundant being Kv5.1. We found that Kv5.1 co-immunoprecipitates with Kv2.1 and to a lesser extent with Kv2.2 from brain lysates, and that Kv5.1 protein levels are decreased by 70% in Kv2.1 knockout mice and 95% in Kv2.1/Kv2.2 double knockout mice. RNAscope and immunolabelling revealed that Kv5.1 is prominently expressed in neocortex, where it is detected in a substantial fraction of Kv2.1/Kv2.2 positive neurons in layers 2/3, 5, and 6. At the subcellular level, Kv5.1 protein is co-clustered with Kv2.1 and Kv2.2 at presumptive ER-PM junctions on the soma and proximal dendrites of cortical neurons. Moreover, in addition to modifying channel conductance, we found that Kv2/Kv5.1 channels are less phosphorylated and insensitive to RY785, a potent and selective Kv2 channel inhibitor. Together, these findings demonstrate that KvS subunits create multiple Kv2 channel subtypes in brain. Most notably, Kv2/Kv5.1 channels are highly expressed in cortical neurons, where their unique properties likely modulate the critical conducting and non-conducting roles of Kv2 channels.Significance Statement Voltage-gated Kv2 potassium channels play important roles in regulating neuronal excitability and organizing endoplasmic reticulum - plasma membrane (ER-PM) junctions in brain neurons. Here, we use mass spectrometry to identify five KvS channel subunits as components of native Kv2 channels in brain. The most abundant of these subunits, Kv5.1, is prominently expressed in cortex, where it clusters at ER-PM junctions with Kv2 subunits in a subpopulation of cortical neurons. Kv5.1 expression depends on Kv2 subunits, and Kv2/Kv5.1 heteromeric channels differ in their biophysical and pharmacological properties. We propose that differential expression of Kv5.1 in subclasses of cortical neurons diversifies Kv2 channel function.