Competition of calcified calmodulin N lobe and PIP₂to an LQT mutation site in Kv7.1 channel

Abstract

Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current I KS that repolarizes the cardiac action potential. The physiological importance of the I KS channel is underscored by the existence of mutations in human Kv7.1 and KCNE1 genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ), but the role of CaM in channel function is still unclear, and its possible interaction with PIP 2 is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP 2 and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1

Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv7.2 potassium channel mutants

“…The low concentration and the GST tag help prevent protein aggregation, and, with the use of this very sensitive assay, quantitative data for the interaction was obtained [10,36,37]. Dose–response binding curves were constructed titrating D-CaM fluorescence after adding increasing concentrations of GST-fusion, in the absence (10 mM EGTA added; Figure 4(c) right ) or in the presence of a physiologically relevant concentration of Ca 2+ (3.9 μM free Ca 2+ ; Figure 4(c) left ).…”

“…It seems unlikely that single channel conductance is affected by a residue that is not even in contact with the internal face of the pore, as revealed by the structure of the homologous Kv7.1 channel [38]. However, this residue is part of a cluster that affects the interaction with PIP 2 in Kv7.1 channels [10], which should alter P open . To assess the impact of PIP 2 sensitivity, the channel was co-expressed in HEK293T cells with a Danio rerio voltage-dependent phosphatase (Dr-VSP).…”

“…Often, facilitation is manifested by a left shift of the voltage-current relationship, such as that caused by apo-CaM for Kv7.2 and Kv7.4 channels [7,9,42], or holo-CaM for Kv7.1 [10]. However, changes in these parameters were not observed (Kv7.2/Kv7.3 heteromers) or were in the opposite direction (Kv7.2 homomers) [27].…”

“…An increase in PIP 2 binding affinity, which is an essential co-factor for Kv7 channel function, can also be discarded as the compensatory mechanism. A PIP 2 binding site has been located at the tri-lysine cluster ( 526 KKK 528 ) of helix B in Kv7.1 [10], which is equivalent to the Kv7.2 524 KRK 526 basic cluster. The corresponding Kv7.1 K528 residue is not anticipated to interact with PIP 2 according to docking and MS simulations, and the equivalent K528N mutant had little or no impact in the interaction with PIP 2 in vitro [10].…”

“…CaM affects not only PIP 2 sensitivity [7,8], but also influences voltage sensitivity [7,9,10], regulation of the stability of the distal tetramerization domain [11], functional connection between this distal coiled-coil tetramerization domain and PIP 2 modulation [12] and plays a role in the suppression of Kv7.2/Kv7.3 currents mediated by bradykinin [13,14]. Furthermore, CaM plays a crucial role in Kv7.2 channel trafficking [15], therefore regulating polarized axonal surface expression [16,17] and the intracellular transport of Kv7.2 proteins to the plasma membrane [18].…”