Phosphorylation of Kv7.2 potassium channel’s regulatory domain alters binding interactions with calmodulin

Title : Phosphorylation of Kv7.2 potassium channel’s regulatory domain alters binding interactions with calmodulin

Abstract:

Kv7 channels are potassium ion channels expressed in the nervous system. Kv7 channels function to control the excitability of neurons by reducing the rate of action potential firing. The Kv7.2 channel has been shown to play a particularly crucial role in neurodevelopment. Five different KCNQ genes encode Kv7 subunits, which form a tetrameric Kv7 channel. Loss of function mutations in the KCNQ2 gene have been shown to result in neurologic dysfunction, including neonatal epilepsy. Therefore, regulation of Kv7.2 is essential for normal neuronal function. Calmodulin (CaM) is a calcium (Ca²⁺)-binding protein shown to interact with the C-terminus of Kv7.2, particularly with its intracellular regulatory domain. This region contains several hotspots for pathological mutations, suggesting their importance in regulating the function of Kv7.2. The regulatory domain of KCNQ2 is composed of two regions of importance to this study: the A helix (Q2A) and B helix (Q2B) (Fig. 1 & 2). CaM binds to each Kv7 subunit by interacting with Q2A and Q2B. The amino acid sequence of Q2B contains phosphorylation sites at serines S520 and S527 which could potentially contribute to the regulation of Kv7.2 function. This study aims to characterize the Ca²⁺-dependent binding interactions between CaM and the regulatory domain of Kv7.2. It is also investigating whether phosphorylation of Q2B interferes with CaM interactions and how CaM modifies its binding to the regulatory domain. Binding interactions were studied by electrophoretic mobility shift assay (EMSA) and isothermal calorimetry (ITC). Results from the ITC show that CaM has a higher affinity for Q2B (Kd = 0.243 μM) than Q2A (Kd = 5.27 μM). Compared to the binding affinity between CaM and an extended Q2B sequence (Kd = 0.05 μM), the binding affinity is 42x lower when Q2B is phosphorylated at S520 (Kd = 2.1 μM) and only 5x lower when phosphorylated at S527 (Kd = 0.25 μM). EMSA shows a degree of CaM binding to Q2B phosphorylated at S527 but demonstrates no binding when phosphorylated at S520. These findings indicate that Q2B phosphorylation at S520 blocks CaM interactions in the presence of Ca²⁺.

Biography:

Sinchana Basoor is a current second-year medical student at Long School of Medicine at UT Health San Antonio. She completed her undergraduate education at Baylor University in 2023, where she defended her thesis on mitochondrial morphology in the pathophysiology of hearing loss. She is currently involved in biochemical research with Dr. Crystal Archer at UT Health San Antonio. She is also conducting clinical research on orthopedic and plastic surgery outcomes in conjunction with University Hospital and the VA Hospital in San Antonio.