Biology subjects

Vierra, N. C.

Publications and source records attributed to Vierra, N. C..

4 recordsLinked to original sources

bioRxiv2024

Electrically silent KvS subunits associate with native Kv2 channels in brain and impact diverse properties of channel function

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 the two pore-forming alpha subunits Kv2.1 and Kv2.2 assemble with "electrically silent" KvS subunits to form heterotetrameric channels with distinct biophysical properties. Here, using mass spectrometry-based proteomics, we identified five KvS subunits as components of native Kv2.1 channels immunopurified from mouse brain, 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 Kv2.1/2.2 double knockout mice. Multiplex immunofluorescent labelling of rodent brain sections revealed that in neocortex Kv5.1 immunolabeling is apparent in a large percentage of Kv2.1 and Kv2.2-positive layer 2/3 neurons, and in a smaller percentage of layer 5 and 6 neurons. At the subcellular level, Kv5.1 is co-clustered with Kv2.1 and Kv2.2 at ER-PM junctions in cortical neurons, although clustering of Kv5.1-containing channels is reduced relative to homomeric Kv2 channels. We also found that in heterologous cells coexpression with Kv5.1 reduces the clustering and alters the pharmacological properties of Kv2.1 channels. Together, these findings demonstrate that the Kv5.1 electrically silent subunit is a component of a substantial fraction of native brain Kv2 channels, and that its incorporation into heteromeric channels can impact diverse aspects of Kv2 channel function.

neuroscience↗

bioRxiv2023

The formation of KV2.1 macro-clusters is required for sex-specific differences in L-type CaV1.2 clustering and function in arterial myocytes

In arterial myocytes, the canonical function of voltage-gated CaV1.2 and KV2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, KV2.1 also plays a sex-specific role by promoting the clustering and activity of CaV1.2 channels. However, the impact of KV2.1 protein organization on CaV1.2 function remains poorly understood. We discovered that KV2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of KV2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the KV2.1 clustering site (KV2.1S590A) eliminated KV2.1 macro-clustering and sex-specific differences in CaV1.2 cluster size and activity. We propose that the degree of KV2.1 clustering tunes CaV1.2 channel function in a sex-specific manner in arterial myocytes.

physiology↗

bioRxiv2019

Epilepsy and neurobehavioral abnormalities in mice with a KCNB1 pathogenic variant that alters conducting and non-conducting functions of KV2.1

Developmental and epileptic encephalopathies (DEE) are a group of severe epilepsies that usually present with intractable seizures, developmental delay and are at a higher risk for premature mortality. Numerous genes have been identified as a monogenic cause of DEE, including KCNB1. The voltage-gated potassium channel KV2.1, encoded by KCNB1, is primarily responsible for delayed rectifier potassium currents that are important regulators of excitability in electrically excitable cells, including neurons and cardiomyocytes. The de novo pathogenic variant KCNB1-p.G379R was identified in an infant with epileptic spasms, atonic, focal and tonic-clonic seizures that were refractory to treatment with standard antiepileptic drugs. Previous work demonstrated deficits in potassium conductance, but did not assess non-conducting functions. To determine if the G379R variant affected clustering at endoplasmic reticulum-plasma membrane junctions KV2.1-G379R was expressed in HEK293T cells. KV2.1-G379R expression did not induce formation of endoplasmic reticulum-plasma membrane junctions, and co-expression of KV2.1-G379R with KV2.1-WT lowered induction of these structures relative to KV2.1-WT alone, suggesting a dominant negative effect. To model this variant in vivo, we introduced Kcnb1G379R into mice using CRISPR/Cas9 genome editing. We characterized neurological and neurobehavioral phenotypes of Kcnb1G379R/+ (Kcnb1R/+) and Kcnb1G379R/G379R (Kcnb1R/R) mice, and screened for cardiac abnormalities. Immunohistochemistry studies on brains from Kcnb1+/+ (WT), Kcnb1R/+ and Kcnb1R/R mice revealed genotype-dependent differences in the levels and subcellular localization of KV2.1, with reduced plasma membrane expression of the KV2.1-G379R protein, consistent with in vitro data. Kcnb1R/+ and Kcnb1R/R mice displayed profound hyperactivity, repetitive behaviors, impulsivity and reduced anxiety. In addition, both Kcnb1R/+ and Kcnb1R/R mice exhibited abnormal interictal EEG abnormalities, including isolated spike and slow waves. Spontaneous seizure events were observed in Kcnb1R/R mice during exposure to novel environments and/or handling, while both Kcnb1R/+ and Kcnb1R/R mutants were more susceptible to induced seizures. Kcnb1R/+ and Kcnb1R/R mice exhibited prolonged rate-corrected QT interval on surface ECG recording. Overall, the Kcnb1G379R mice recapitulate many features observed in individuals with DEE due to pathogenic variants in KCNB1. This new mouse model of KCNB1 associated DEE will be valuable for improving the understanding of the underlying pathophysiology and will provide a valuable tool for the development of therapies to treat this pharmacoresistant DEE.

neuroscience↗

bioRxiv2019

Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in neurons

The voltage-gated K+ channel Kv2.1 serves a major structural role in the soma and proximal dendrites of brain neurons, tethering the plasma membrane (PM) to the endoplasmic reticulum (ER). Although Kv2.1 clustering at neuronal ER-PM junctions (EPJs) is tightly regulated and conserved across species, its function at these sites is unclear. By identifying and evaluating proteins in close spatial proximity to Kv2.1-containing EPJs, we discovered that a significant role of Kv2.1 at EPJs is to promote the clustering and functional coupling of PM L-type Ca2+ channels (LTCCs) to ryanodine receptor (RyR) ER Ca2+ release channels. Kv2.1 clustering also unexpectedly enhanced LTCC opening at polarized membrane potentials. This enabled Kv2.1-LTCC-RyR triads to generate localized Ca2+ release events (i.e., Ca2+ sparks) independently of action potentials. Together, these findings uncover a novel mode of LTCC regulation and establish a unique mechanism whereby Kv2.1-associated EPJs provide a molecular platform for localized somatodendritic Ca2+ signals.

neuroscience↗