A unique mechanism of inactivation gating of the Kv channel family member Kv7.1 and its modulation by PIP2 and calmodulin.

Inactivation of voltage-gated K+ (Kv) channels mostly occurs by fast N-type or/and slow C-type mechanisms. Here, we characterized a unique mechanism of inactivation gating comprising two inactivation states in a member of the Kv channel superfamily, Kv7.1. Removal of external Ca2+ in wild-type Kv7.1 channels produced a large, voltage-dependent inactivation, which differed from N- or C-type mechanisms. Glu295 and Asp317 located, respectively, in the turret and pore entrance are involved in Ca2+ coordination, allowing Asp317 to form H-bonding with the pore helix Trp304, which stabilizes the selectivity filter and prevents inactivation. Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+-calmodulin prevented Kv7.1 inactivation triggered by Ca2+-free external solutions, where Ser182 at the S2-S3 linker relays the calmodulin signal from its inner boundary to the external pore to allow proper channel conduction. Thus, we revealed a unique mechanism of inactivation gating in Kv7.1, exquisitely controlled by external Ca2+ and allosterically coupled by internal PIP2 and Ca2+-calmodulin.

Ca2+-Calmodulin and PIP2 interactions at the proximal C-terminus of Kv7 channels.

In the heart, co-assembly of Kv7.1 with KCNE1 produces the slow IKS potassium current, which repolarizes the cardiac action potential and mutations in human Kv7.1 and KCNE1 genes cause cardiac arrhythmias. The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2) and recently we revealed the competition of PIP2 with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation. Data indicated that PIP2 and Ca2+-CaM perform the same function on IKS channel gating to stabilize the channel open state. Here we show that similar features were observed for Kv7.1 currents expressed alone. We also find that conservation of homologous residues in helix B of other Kv7 subtypes confer similar competition of Ca2+-CaM with PIP2 binding to their proximal C-termini and suggest that PIP2-CaM interactions converge to Kv7 helix B to modulates channel activity in a Kv7 subtype-dependent manner.

Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel.

Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current IKS that repolarizes the cardiac action potential. The physiological importance of the IKS 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 (PIP2), but the role of CaM in channel function is still unclear, and its possible interaction with PIP2 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 PIP2 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 helix B form a critical site where CaM competes with PIP2 to stabilize the channel open state. Data indicate that both PIP2 and Ca2+-CaM perform the same function on IKS channel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca2+-CaM. The results suggest that, after receptor-mediated PIP2 depletion and increased cytosolic Ca2+, calcified CaM N lobe interacts with helix B in place of PIP2 to limit excessive IKS current inhibition.

Formation of halogenated acetaldehydes, and occurrence in Canadian drinking water.

Controlled laboratory chlorination of acetaldehyde (ACD) under typical drinking water conditions (pH 6.7, 7.6 and 8.8, and temperature 4 degrees C and 21 degrees C) revealed that the formation of chloral hydrate (CH), the most common halogenated acetaldehyde (HAs), increased with contact time (0-10 days). However, at increased pH and temperature, CH reached maximum levels and subsequently broke down partially to chloroform and other unidentified compounds. After 10 days contact time, a maximum of 63% (molar) of the initial ACD consumed were converted into CH or chloroform (TCM). Various surveys of drinking water systems indicated that ACD is not the only precursor of CH. A suite of aldehydes (including ACD), and chlorinated disinfection by-products (including TCM and CH) were found in most distribution systems. The levels of bromide in source water impacted speciation of HAs. In addition to CH, brominated and other mixed (Cl/Br) acetaldehydes were detected in most samples; the speciation of HAs and THMs followed comparable trends. Similar to chloroform for trihalomethanes, CH contributed from as low as 5% to up to 60% of the total HAs. The bromine incorporation factors (BIF) in THMs and HAs were shown to increase with increasing bromide ion concentrations in the source water. Brominated THMs are more readily formed than their HA analogues; in fact, BIF values for THMs were 2-3 times higher than for the HAs. It was found that HAs may be as high as THMs in some drinking waters. As a result, the determination of the other target HAs, in addition to CH, is necessary for a better assessment of the pool of disinfection by-products in drinking water.

Halogenated acetaldehydes: analysis, stability and fate in drinking water.

In our previous studies, chloral hydrate has been the only chlorinated acetaldehyde determined in drinking water because authentic standards of other related haloacetaldehydes were not available. Recently, standards of dichloroacetaldehyde, bromochloroacetaldehyde, dibromoacetaldehyde, bromodichloroacetaldehyde, chlorodibromoacetaldehyde, and tribromoacetaldehyde have become available commercially. They were obtained and verified for purity and stability using a dual-column GC-ECD system. Each commercial standard was found to contain small amounts of the other target haloacetaldehydes (HAs). The stability of the HAs stock solutions was solvent dependent: in acetone, the brominated species partially degraded to bromoacetone, while all target HAs were stable in MTBE for up to 8 months. The analytical parameters, required for the quantification of HAs in water, were determined and used to evaluate the stability of the HAs in water. Under the conditions of the sampling protocol (field pH adjustment to pH 4.5 and storage at 4 degrees C), the target HAs were stable in water for up to 14 days. However, at typical drinking water pH and temperature conditions, the stability varied with the HA species, pH, temperature and storage period. The trihalogenated acetaldehydes degraded, in part, to their corresponding trihalomethanes (THMs) at increasing pH and temperature. Most target HAs were detected in drinking water samples collected from various Canadian drinking water systems, and the speciation was dependent on water parameters (e.g. bromide concentration) and treatment processes. From the water samples analysed, chloral hydrate ranged between 7% and 51% of the total HAs (w/w). The weigh ratio of total HAs to total THMs (10-46%) indicated that HAs contributed significantly to the pool of DBPs in drinking water.