Marc Borsotto

Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype

Depression is a devastating illness with a lifetime prevalence of up to 20%. The neurotransmitter serotonin or 5-hydroxytryptamine (5-HT) is involved in the pathophysiology of depression and in the effects of antidepressant treatments. However, molecular alterations that underlie the pathology or treatment of depression are still poorly understood. The TREK-1 protein is a background K+ channel regulated by various neurotransmitters including 5-HT. In mice, the deletion of its gene (Kcnk2, also called TREK-1) led to animals with an increased efficacy of 5-HT neurotransmission and a resistance to depression in five different models and a substantially reduced elevation of corticosterone levels under stress. TREK-1–deficient (Kcnk2−/−) mice showed behavior similar to that of naive animals treated with classical antidepressants such as fluoxetine. Our results indicate that alterations in the functioning, regulation or both of the TREK-1 channel may alter mood, and that this particular K+ channel may be a potential target for new antidepressants.

KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel

Mutations in HERG and KCNQ1 (or KVLQT1) genes cause the life‐threatening Long QT syndrome. These genes encode K+ channel pore‐forming subunits that associate with ancillary subunits from the KCNE family to underlie the two components, IKr and IKs, of the human cardiac delayed rectifier current IK. The KCNE family comprises at least three members. KCNE1 (IsK or MinK) recapitulates IKs when associated with KCNQ1, whereas it augments the amplitude of an IKr‐like current when co‐expressed with HERG. KCNE3 markedly changes KCNQ1 as well as HERG current properties. So far, KCNE2 (MirP1) has only been shown to modulate HERG current. Here we demonstrate the interaction of KCNE2 with the KCNQ1 subunit, which results in a drastic change of KCNQ1 current amplitude and gating properties. Furthermore, KCNE2 mutations also reveal their specific functional consequences on KCNQ1 currents. KCNQ1 and HERG appear to share unique interactions with KCNE1, 2 and 3 subunits. With the exception of KCNE3, mutations in all these partner subunits have been found to lead to an increased propensity for cardiac arrhythmias.

p11, an annexin II subunit, an auxiliary protein associated with the background K+ channel, TASK-1

TASK‐1 belongs to the 2P domain K+ channel family and is the prototype of background K+ channels that set the resting membrane potential and tune action potential duration. Its activity is highly regulated by hormones and neurotransmitters. Although numerous auxiliary proteins have been described to modify biophysical, pharmacological and expression properties of different voltage‐ and Ca2+‐sensitive K+ channels, none of them is known to modulate 2P domain K+ channel activity. We show here that p11 interacts specifically with the TASK‐1 K+ channel. p11 is a subunit of annexin II, a cytoplasmic protein thought to bind and organize specialized membrane cytoskeleton compartments. This association with p11 requires the integrity of the last three C‐terminal amino acids, Ser‐Ser‐Val, in TASK‐1. Using series of C‐terminal TASK‐1 deletion mutants and several TASK‐1–GFP chimeras, we demonstrate that association with p11 is essential for trafficking of TASK‐1 to the plasma membrane. p11 association with the TASK‐1 channel masks an endoplasmic reticulum retention signal identified as Lys‐Arg‐Arg that precedes the Ser‐Ser‐Val sequence.

The KCNQ2 potassium channel: splice variants, functional and developmental expression. Brain localization and comparison with KCNQ3

Benign familial neonatal convulsions, an autosomal dominant epilepsy of newborns, are linked to mutations affecting two six‐transmembrane potassium channels, KCNQ2 and KCNQ3. We isolated four splice variants of KCNQ2 in human brain. Two forms generate, after transient expression in COS cells, a potassium‐selective current similar to the KCNQ1 current. L‐735,821, a benzodiazepine molecule which inhibits the KCNQ1 channel activity (EC50=0.08 μM), also blocks KCNQ2 currents (EC50=1.5 μM). Using in situ hybridization, KCNQ2 and KCNQ3 have been localized within the central nervous system, in which they are expressed in the same areas, mainly in the hippocampus, the neocortex and the cerebellar cortex. During brain development, KCNQ3 is expressed later than KCNQ2.

Purification of the dihydropyridine receptor of the voltage-dependent Ca2+ channel from skeletal muscle transverse tubules using (+) [3H]PN 200-110

The rabbit skeletal muscle T-tubule membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[3H]PN 200-110 binding experiments. The dissociation constant of the (+) [3H]PN 200-110 solubilized-receptor complex was 0.4 +/- 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (k1 = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 10(-2) min-1). The (+) [3H]PN 200-110 receptor has been substantially purified by a combination of filtration of Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [3H]PN 200-110. The purified material contained polypeptides of apparent molecular weights of 142 000, 32 000 and 33 000. These three components copurified with (+)[3H]PN 200-110 binding activity.

Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design

We found that spadin, a natural peptide derived from sortilin, blocks the mouse TREK-1 channel and might be an efficient and fast-acting antidepressant.

M-type KCNQ2-KCNQ3 potassium channels are modulated by the KCNE2 subunit.

KCNQ2 and KCNQ3 subunits belong to the six transmembrane domain K+ channel family and loss of function mutations are associated with benign familial neonatal convulsions. KCNE2 (MirP1) is a single transmembrane domain subunit first described to be a modulator of the HERG potassium channel in the heart. Here, we show that KCNE2 is present in brain, in areas which also express KCNQ2 and KCNQ3 channels. We demonstrate that KCNE2 associates with KCNQ2 and/or KCNQ3 subunits. In transiently transfected COS cells, KCNE2 expression produces an acceleration of deactivation kinetics of KCNQ2 and of the KCNQ2–KCNQ3 complex. Effects of two previously identified arrhythmogenic mutations of KCNE2 have also been analyzed.

ATP-sensitive potassium channels (KATP) in retina: a key role for delayed ischemic tolerance

The objectives of the present study were to determine the localization of K(ATP) channels in normal retina and to evaluate their potential roles in ischemic preconditioning (IPC) in a rat model of ischemia induced by increased intraocular pressure (IOP). Brown Norway rats were subjected to sublethal 3-, lethal 20- and 40-min ischemia and the functional recovery was evaluated using electroretinography. The time interval between ischemic insults ranged from 1 to 72 h. The effects of K(ATP) channel blockade on IPC protection were studied by treatment with 0.01% glipizide. IPC was mimicked by injection of K(ATP) channel openers of 0.01% (-)cromakalim or 0.01% P1060 72 h before 20-min ischemia. Co-expression of K(ATP) channel subunits Kir6.2/SUR1 was observed in the retinal pigment epithelium, inner segments of photoreceptors, outer plexiform and ganglion cell layers and at the border of the inner nuclear layer. In contrast to a 20- or 40-min ischemia, a 3-min ischemia induced no alteration of the electroretinogram (ERG) and constituted the preconditioning stimulus. An ischemic challenge of 40 min in preconditioned rats induced impairment of retinal function. However, animals preconditioned 24, 48 and 72 h before 20-min ischemia had a significant improvement of the ERG. (-)Cromakalim and P1060 mimicked the effect of IPC. Glipizide significantly suppressed the protective effects of preconditioning. In conclusion, activation of K(ATP) channels plays an important role in the mechanism of preconditioning by enhancing the resistance of the retina against a severe ischemic insult.

Neuroprotective and neuroproliferative activities of NeuroAid (MLC601, MLC901), a Chinese medicine, in vitro and in vivo

Although stroke remains a leading cause of death and adult disability, numerous recent failures in clinical stroke trials have led to some pessimism in the field. Interestingly, NeuroAid (MLC601), a traditional medicine, particularly used in China, South East Asia and Middle East has been reported to have beneficial effects in patients, particularly in post-stroke complications. Here, we demonstrate in a rodent model of focal ischemia that NeuroAid II (MLC901) pre- and post-treatments up to 3 h after stroke improve survival, protect the brain from the ischemic injury and drastically decrease functional deficits. MLC601 and MLC901 also prevent neuronal death in an in vitro model of excitotoxicity using primary cultures of cortical neurons exposed to glutamate. In addition, MLC601/MLC901 treatments were shown to induce neurogenesis in rodent and human cells, promote cell proliferation as well as neurite outgrowth and stimulate the development of a dense axonal and dendritic network. MLC601 and MLC901 clearly represent a very interesting strategy for stroke treatment at different stages of the disease.

Determination of the molecular size of the nitrendipine-sensitive Ca2+ channel by radiation inactivation.

The molecular size of the [3H] nitrendipine receptor of transverse tubules prepared from rabbit skeletal muscle and from rat cortex synaptic membranes have been investigated. Radiation inactivation of the specific binding of [3H] nitrendipine was consistent with Mr equals 210 000 +/- 20,000 for the receptor in each membrane preparation indicating a common size for the [3H] nitrendipine receptor.