Drosophila KCNQ Channel Displays Evolutionarily Conserved Electrophysiology and Pharmacology with Mammalian KCNQ Channels

Of the five human KCNQ (Kv7) channels, KCNQ1 with auxiliary subunit KCNE1 mediates the native cardiac IKs current with mutations causing short and long QT cardiac arrhythmias. KCNQ4 mutations cause deafness. KCNQ2/3 channels form the native M-current controlling excitability of most neurons, with mutations causing benign neonatal febrile convulsions. Drosophila contains a single KCNQ (dKCNQ) that appears to serve alone the functions of all the duplicated mammalian neuronal and cardiac KCNQ channels sharing roughly 50–60% amino acid identity therefore offering a route to investigate these channels. Current information about the functional properties of dKCNQ is lacking therefore we have investigated these properties here. Using whole cell patch clamp electrophysiology we compare the biophysical and pharmacological properties of dKCNQ with the mammalian neuronal and cardiac KCNQ channels expressed in HEK cells. We show that Drosophila KCNQ (dKCNQ) is a slowly activating and slowly-deactivating K+ current open at sub-threshold potentials that has similar properties to neuronal KCNQ2/3 with some features of the cardiac KCNQ1/KCNE1 accompanied by conserved sensitivity to a number of clinically relevant KCNQ blockers (chromanol 293B, XE991, linopirdine) and opener (zinc pyrithione). We also investigate the molecular basis of the differential selectivity of KCNQ channels to the opener retigabine and show a single amino acid substitution (M217W) can confer sensitivity to dKCNQ. We show dKCNQ has similar electrophysiological and pharmacological properties as the mammalian KCNQ channels, allowing future study of physiological and pathological roles of KCNQ in Drosophila and whole organism screening for new modulators of KCNQ channelopathies

Synergistic interplay of Gβγ and phosphatidylinositol 4,5-bisphosphate dictates Kv7.4 channel activity.

Kv7.4 channels are key determinants of arterial contractility and cochlear mechanosensation that, like all Kv7 channels, have an obligatory requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). βγ G proteins (Gβγ) have been identified as novel positive regulators of Kv7.4. The present study ascertained whether Gβγ increased Kv7.4 open probability through an increased sensitivity to PIP2. In HEK cells stably expressing Kv7.4, PIP2 or Gβγ increased open probability in a concentration dependent manner. Depleting PIP2 prevented any Gβγ-mediated stimulation whilst an array of Gβγ inhibitors prohibited any PIP2-induced current enhancement. A combination of PIP2 and Gβγ at sub-efficacious concentrations increased channel open probability considerably. The stimulatory effects of three Kv7.2-7.5 channel activators were also lost by PIP2 depletion or Gβγ inhibitors. This study alters substantially our understanding of the fundamental processes that dictate Kv7.4 activity, revealing a more complex and subtle paradigm where the reliance on local phosphoinositide is dictated by interaction with Gβγ

KCNQ2 encephalopathy: Emerging phenotype of a neonatal epileptic encephalopathy

ObjectiveKCNQ2 and KCNQ3 mutations are known to be responsible for benign familial neonatal seizures (BFNS). A few reports on patients with a KCNQ2 mutation with a more severe outcome exist, but a definite relationship has not been established. In this study we investigated whether KCNQ2/3 mutations are a frequent cause of epileptic encephalopathies with an early onset and whether a recognizable phenotype exists.MethodsWe analyzed 80 patients with unexplained neonatal or early-infantile seizures and associated psychomotor retardation for KCNQ2 and KCNQ3 mutations. Clinical and imaging data were reviewed in detail.ResultsWe found 7 different heterozygous KCNQ2 mutations in 8 patients (8/80; 10%); 6 mutations arose de novo. One parent with a milder phenotype was mosaic for the mutation. No KCNQ3 mutations were found. The 8 patients had onset of intractable seizures in the first week of life with a prominent tonic component. Seizures generally resolved by age 3 years but the children had profound, or less frequently severe, intellectual disability with motor impairment. Electroencephalography (EEG) at onset showed a burst-suppression pattern or multifocal epileptiform activity. Early magnetic resonance imaging (MRI) of the brain showed characteristic hyperintensities in the basal ganglia and thalamus that later resolved.InterpretationKCNQ2 mutations are found in a substantial proportion of patients with a neonatal epileptic encephalopathy with a potentially recognizable electroclinical and radiological phenotype. This suggests that KCNQ2 screening should be included in the diagnostic workup of refractory neonatal seizures of unknown origin.Sarah Weckhuysen... Sarah E. Heron, John C. Mulley... et al

Bimodal effects of the Kv7 channel activator retigabine on vascular K+ currents

Yeung SYM, Schwake M, Pucovský V, Greenwood IA. Bimodal effects of the K 7 channel activator retigabine on vascular K+ currents. British Journal of Pharmacology. 2008;155(1):62-72.Background and purpose: This study investigated the functional and electrophysiological effects of the Kv7 channel activator, retigabine, on murine portal vein smooth muscle. Experimental approach: KCNQ gene expression was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemical experiments. Whole cell voltage clamp and current clamp were performed on isolated myocytes from murine portal vein. Isometric tension recordings were performed on whole portal veins. K+ currents generated by KCNQ4 and KCNQ5 expression were recorded by two-electrode voltage clamp in Xenopus oocytes. Key results: KCNQ1, 4 and 5 were expressed in mRNA derived from murine portal vein, either as whole tissue or isolated myocytes. Kv7.1 and Kv7.4 proteins were identified in the cell membranes of myocytes by immunocytochemistry. Retigabine (2–20 μM) suppressed spontaneous contractions in whole portal veins, hyperpolarized the membrane potential and augmented potassium currents at −20 mV. At more depolarized potentials, retigabine and flupirtine, decreased potassium currents. Both effects of retigabine were prevented by prior application of the Kv7 blocker XE991 (10 μM). Recombinant KCNQ 4 or 5 channels were only activated by retigabine or flupirtine. Conclusions and implications: The Kv7 channel activators retigabine and flupirtine have bimodal effects on vascular potassium currents, which are not seen with recombinant KCNQ channels. These results provide support for KCNQ4- or KCNQ5-encoded channels having an important functional impact in the vasculature

Molecular expression and pharmacological identification of a role for Kv7 channels in murine vascular reactivity

BACKGROUND AND PURPOSE: This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility. EXPERIMENTAL APPROACH: Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta. KEY RESULTS: All blood vessels expressed KCNQ1, 4 and 5 with hitherto ‘neuronal' KCNQ4 being, surprisingly, the most abundant. The correlated proteins K(v)7.1, K(v)7.4 and K(v)7.5 were identified in the cell membranes of aortic myocytes by immunocytochemistry. Application of three compounds known to activate K(v)7 channels, retigabine (2 –20 μM), flupirtine (20 μM) and meclofenamic acid (20 μM), relaxed vessels precontracted by phenylephrine or 1 mM 4-aminopyridine but had no effect on contractions produced by 60 mM KCl or the K(v)7 channel blocker XE991 (10 μM). All vessels tested contracted upon application of the K(v)7 channel blockers XE991 and linopirdine (0.1-10 μM). CONCLUSIONS AND IMPLICATIONS: Murine blood vessels exhibit a distinctive KCNQ expression profile with ‘neuronal' KCNQ4 dominating. The ion channels encoded by KCNQ genes have a crucial role in defining vascular reactivity as K(v)7 channel blockers produced marked contractions whereas K(v)7 channel activators were effective vasorelaxants