The Traditional Chinese Medicine MLC901 inhibits inflammation processes after focal cerebral ischemia.

Inflammation is considered as a major contributor to brain injury following cerebral ischemia. The therapeutic potential of both MLC601/MLC901, which are herbal extract preparations derived from Chinese Medicine, has been reported both in advanced stroke clinical trials and also in animal and cellular models. The aim of this study was to investigate the effects of MLC901 on the different steps of post-ischemic inflammation in focal ischemia in mice. In vivo injury was induced by 60 minutes of middle cerebral artery occlusion (MCAO) followed by reperfusion. MLC901 was administered in post-treatment 90 min after the onset of ischemia and once a day during reperfusion. MLC901 treatment resulted in a reduction in infarct volume, a decrease of Blood Brain Barrier leakage and brain swelling, an improvement in neurological scores and a reduction of mortality rate at 24 hours after MCAO. These beneficial effects of MLC901 were accompanied by an inhibition of astrocytes and microglia/macrophage activation, a drastically decreased neutrophil invasion into the ischemic brain as well as by a negative regulation of pro-inflammatory mediator expression (cytokines, chemokines, matrix metalloproteinases). MLC901 significantly inhibited the expression of Prx6 as well as the transcriptional activity of NFκB and the activation of Toll-like receptor 4 (TLR4) signaling, an important pathway in the immune response in the ischemic brain. MLC901 effects on the neuroinflammation cascade induced by cerebral ischemia probably contribute, in a very significant way, in its potential therapeutic value.

Positive effects of the traditional Chinese medicine MLC901 in cognitive tasks.

MLC901 (NurAiDII) is used as a treatment for stroke patients. It has been shown that MLC901 improves motor and cognitive recovery in ischemic and traumatic brain-injured rodents. The present study seeks to delineate cognitive effects induced by MLC901 in normal, noninjured mice. To this end, the behaviors of vehicle- and MLC901-treated C57BL/6 mice in hippocampus-dependent (passive avoidance, Morris water maze) and hippocampus-independent (novel object recognition) cognitive tasks are compared. The potential influence of the compound on the anxiety level and nycthemeral rhythm of mice is also assessed. In addition, the long-term effects of MLC901 on hippocampal neurogenesis are measured. The results clearly demonstrate that MLC901 promotes extinction in passive avoidance and reversal learning in the Morris water maze and improves the performance of mice in novel object recognition. In parallel, this study shows the long-term proneurogenesis effects of MLC901 that result in the increase in the number of mature neurons in the hippocampus. If these observations can be extended to humans, then MLC901 could represent a promising therapeutic strategy.

MLC901, a Traditional Chinese Medicine induces neuroprotective and neuroregenerative benefits after traumatic brain injury in rats.

Traumatic brain injury (TBI) is a frequent and clinically highly heterogeneous neurological disorder with large socioeconomic consequences. NeuroAid (MLC601 and MLC901), a Traditional Medicine used in China for patients after stroke has been previously reported to induce neuroprotection and neuroplasticity. This study was designed to evaluate the neuroprotective and neuroregenerative effects of MLC901 in a rat model of TBI. TBI was induced by a moderate lateral fluid percussion applied to the right parietal cortex. MLC901 was injected intraperitoneally at 2h post-TBI, and then administered in drinking water at a concentration of 10mg/ml until sacrifice of the animals. The cognitive deficits induced by TBI were followed by using the "what-where-when" task, which allows the measurement of episodic-like memory. MLC901 treatment decreased brain lesions induced by TBI. It prevented the serum increase of S-100 beta (S100B) and neuron-specific enolase (NSE), which may be markers to predict the neurologic outcome in human patients with TBI. MLC901 reduced the infarct volume when injected up to 2h post-TBI, prevented edema formation and assisted its resolution, probably via the regulation of aquaporin 4. These positive MLC901 effects were associated with an upregulation of vascular endothelial growth factor (VEGF) as well as an increase of endogenous hippocampal neurogenesis and gliogenesis around the lesion. Furthermore, MLC901 reduced cognitive deficits induced by TBI. Rats subjected to TBI displayed a suppression of temporal order memory, which was restored by MLC901. This work provides evidence that MLC901 has neuroprotective and neurorestorative actions, which lead to an improvement in the recovery of cognitive functions in a model of traumatic brain injury.

NeuroAiD: properties for neuroprotection and neurorepair.

BACKGROUND: Treatments for stroke and other brain injuries are limited. NeuroAiD has been shown to be beneficial in clinical studies. We reviewed the pharmacological effects of NeuroAiD on the normal and ischemic brain and neurons. METHODS: In vivo and in vitro experiments using mouse model of stroke (focal ischemia), rat model of cardiac arrest (global ischemia) and cortical neurons in culture were reviewed and summarized. RESULTS: NeuroAiD improved survival, attenuated infarct size, improved functional recovery in the model of focal ischemia, and protected neurons against glutamate-induced injury. Furthermore, it enhanced cognitive recovery by reducing hippocampal CA1 cell degeneration, DNA fragmentation, Bax expression and ma-londialdehyde release in the model of global ischemia. Activation of the Akt survival pathway and opening of KATP channels may contribute to the neuroprotective properties of NeuroAiD. NeuroAiD increased BDNF expression and induced proliferation of cells which differentiate and mature into neurons. It enhanced rosette formation of human embryonic stem cells. NeuroAiD-treated embryonic cortical neurons developed into neurons with longer neurites, denser outgrowths and networks, and more synaptic release sites. CONCLUSIONS: NeuroAiD demonstrated both neuroprotective and neuroregenerative properties in rodent models of focal and global ischemia and in cortical cell cultures. These properties would be important for developing a treatment strategy in reducing the long-term disability of stroke, cardiac arrest and other brain injuries.

Activation of ATP-sensitive potassium channels as an element of the neuroprotective effects of the Traditional Chinese Medicine MLC901 against oxygen glucose deprivation.

NeuroAid (MLC601 and MLC901), a Traditional Medicine used in China for patients after stroke has been reported in preclinical models of ischemia to induce neuroprotection and neuroplasticity. This work shows the effects of MLC901 on an in vitro model of oxygen glucose deprivation (OGD). MLC901 prevents neuronal death induced by 120 min OGD and decreases the exaggerated Ca²âº entry in mature cortical neurons exposed to 120 min OGD. The neuroprotective effect of MLC901 is associated with a large hyperpolarization of ∼20 mV which is antagonized by glibenclamide, the specific inhibitor of K(ATP) channels. In addition MLC901 strengthens the activation of K(ATP) channels. MLC901 has been directly shown to act as an activator of K(ATP) channels as potent as the classical K(ATP) channel opener. The capacity of MLC901 to produce a large hyperpolarization, particularly in neurons that have suffered from energy deprivation probably plays an important role in the neuroprotective effects of this traditional medicine that comes in addition to its previously demonstrated neuroregenerative properties.

MLC901, a traditional Chinese medicine protects the brain against global ischemia.

Global ischemia leads to damage in the hippocampal CA1 region and is associated with behavioral deficits. NeuroAid (MLC601 and MLC901), a Traditional Chinese Medicine is used in China for patients after stroke. We have investigated here the effects of MLC901 on brain injury and deficits after global ischemia in the rat. Global ischemia induced by four-vessel occlusion resulted in degeneration of CA1 neurons. MLC901 (0.074 mg/ml) prevented both necrosis and apoptosis of neurons up to 3 h after ischemia. These positive MLC901 effects were associated with a decrease in Bax expression and in levels of the lipid peroxidation product malondialdehyde. Using the PI3-kinase inhibitor LY294002 we also demonstrated the critical role of the Akt pathway in MLC901-mediated neuroprotection. MLC901 enhanced neurogenesis. Furthermore, MLC901 improved functional recovery of rats after global ischemia as assessed by the Morris water maze. In this test MLC901 reduced the increase in escape latency and in swim distance induced by ischemia. MLC901 also improved post-ischemic grip strength. If observations made with rats can be extended to humans, then MLC901 will represent a novel therapeutic strategy after cardiac arrest with a clinically interesting time window of protection.

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.

PP2A-Bgamma subunit and KCNQ2 K+ channels in bipolar disorder.

Many bipolar affective disorder (BD) susceptibility loci have been identified but the molecular mechanisms responsible for the disease remain to be elucidated. In the locus 4p16, several candidate genes were identified but none of them was definitively shown to be associated with BD. In this region, the PPP2R2C gene encodes the Bgamma-regulatory subunit of the protein phosphatase 2A (PP2A-Bgamma). First, we identified, in two different populations, single nucleotide polymorphisms and risk haplotypes for this gene that are associated to BD. Then, we used the Bgamma subunit as bait to screen a human brain cDNA library with the yeast two-hybrid technique. This led us to two new splice variants of KCNQ2 channels and to the KCNQ2 channel itself. This unusual K+ channel has particularly interesting functional properties and belongs to a channel family that is already known to be implicated in several other monogenic diseases. In one of the BD populations, we also found a genetic association between the KCNQ2 gene and BD. We show that KCNQ2 splice variants differ from native channels by their shortened C-terminal sequences and are unique as they are active and exert a dominant-negative effect on KCNQ2 wild-type (wt) channel activity. We also show that the PP2A-Bgamma subunit significantly increases the current generated by KCNQ2wt, a channel normally inhibited by phosphorylation. The kinase glycogen synthase kinase 3 beta (GSK3beta) is considered as an interesting target of lithium, the classical drug used in BD. GSK3beta phosphorylates the KCNQ2 channel and this phosphorylation is decreased by Li+.

Exploration of the pore structure of a peptide-gated Na+ channel.

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.

Cloning of a new mouse two-P domain channel subunit and a human homologue with a unique pore structure.

Mouse KCNK6 is a new subunit belonging to the TWIK channel family. This 335-amino acid polypeptide has four transmembrane segments, two pore-forming domains, and a Ca2+-binding EF-hand motif. Expression of KCNK6 transcripts is principally observed in eyes, lung, stomach and embryo. In the eyes, immunohistochemistry reveals protein expression only in some of the retina neurons. Although KCNK6 is able to dimerize as other functional two-P domain K+ channels when it is expressed in COS-7 cells, it remains in the endoplasmic reticulum and is unable to generate ionic channel activity. Deletions, mutations, and chimera constructions suggest that KCNK6 is not an intracellular channel but rather a subunit that needs to associate with a partner, which remains to be discovered, in order to reach the plasma membrane. A closely related human KCNK7-A subunit has been cloned. KCNK7 displays an intriguing GLE sequence in its filter region instead of the G(Y/F/L)G sequence, which is considered to be the K+ channel signature. This subunit is alternatively spliced and gives rise to the shorter forms KCNK7-B and -C. None of the KCNK7 structures can generate channel activity by itself. The KCNK7 gene is situated on chromosome 11, in the q13 region, where several candidate diseases have been identified.

Expression of TWIK-1, a novel weakly inward rectifying potassium channel in rat kidney.

Several K+ conductances have been identified in the kidney, with specific properties and localization in distinct cell types and membrane domains. On the other hand, several K+ channels have been characterized at the molecular level. By immunolocalization, we show that a new inward rectifying K+ channel, TWIK-1, is specifically expressed in distinct tubular segments and cell types of the rat kidney. In the proximal tubule, TWIK-1 prevails in the initial portions (convoluted part), where it is restricted to the apical (brush-border) membrane. In the collecting duct, immunofluorescence was intracellular or confined to the apical membrane and restricted to intercalated cells, i.e., in cells lacking aquaporin-2, as shown by double immunofluorescence. TWIK was also expressed in medullary and cortical parts of the thick limb of the loop of Henle, identified with an anti-Tamm-Horsfall protein antibody (double immunofluorescence). The intensity of TWIK-1 immunolabeling was unchanged in rats fed a low-Na+ or a low-K+ diet. Because TWIK-1 shares common properties with the low-conductance apical K+ channel of the collecting duct, we propose that it could play a role in K+ secretion, complementary to ROMK, another recently characterized K+ channel located in principal cells of the cortical collecting duct and in the loop of Henle.

Cloning and expression of a novel pH-sensitive two pore domain K+ channel from human kidney.

A complementary DNA encoding a novel K+ channel, called TASK-2, was isolated from human kidney and its gene was mapped to chromosome 6p21. TASK-2 has a low sequence similarity to other two pore domain K+ channels, such as TWIK-1, TREK-1, TASK-1, and TRAAK (18-22% of amino acid identity), but a similar topology consisting of four potential membrane-spanning domains. In transfected cells, TASK-2 produces noninactivating, outwardly rectifying K+ currents with activation potential thresholds that closely follow the K+ equilibrium potential. As for the related TASK-1 and TRAAK channels, the outward rectification is lost at high external K+ concentration. The conductance of TASK-2 was estimated to be 14.5 picosiemens in physiological conditions and 59.9 picosiemens in symmetrical conditions with 155 mM K+. TASK-2 currents are blocked by quinine (IC50 = 22 microM) and quinidine (65% of inhibition at 100 microM) but not by the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine, and Cs+. They are only slightly sensitive to Ba2+, with less than 17% of inhibition at 1 mM. As TASK-1, TASK-2 is highly sensitive to external pH in the physiological range. 10% of the maximum current was recorded at pH 6. 5 and 90% at pH 8.8. Unlike all other cloned channels with two pore-forming domains, TASK-2 is essentially absent in the brain. In human and mouse, TASK-2 is mainly expressed in the kidney, where in situ hybridization shows that it is localized in cortical distal tubules and collecting ducts. This localization, as well as its functional properties, suggest that TASK-2 could play an important role in renal K+ transport.

Genotype-phenotype analysis of a newly discovered family with Liddle's syndrome.

OBJECTIVE: To investigate the clinical, biologic, and molecular abnormalities in a family with Liddle's syndrome and analyze the short- and long-term efficacies of amiloride treatment. PATIENTS: The pedigree consisted of one affected mother and four children, of whom three suffered from early-onset and moderate-to-severe hypertension. METHODS: In addition to the biochemical and hormonal measurements, genetic analysis of the carboxy terminus of the beta subunit of the epithelial sodium channel (beta ENaC) was conducted through single-strand conformation analysis and direct sequencing. The functional properties of the mutation were analyzed using the Xenopus expression system and compared with one mutation affecting the proline-rich sequence of the beta ENaC. RESULTS: Mild hypokalemia and suppressed levels of plasma renin and aldosterone were observed in all affected subjects. Administration of 10 mg/day amiloride for 2 months normalized the blood pressure and plasma potassium levels of all of the affected subjects, whereas their plasma and urinary aldosterone levels remained surprisingly low. A similar pattern was observed after 11 years of follow-up, but a fivefold increase in plasma aldosterone was observed under treatment with 20 mg/day amiloride for 2 weeks. Genetic analysis of the beta ENaC revealed a deletion of 32 nucleotides that had modified the open reading frame and introduced a stop codon at position 582. Expression of this beta 579del32 mutant caused a 3.7 +/- 0.3-fold increase in the amiloride-sensitive sodium current, without modification of the unitary properties of the channel. A similar increase was elicited by one mutation affecting the carboxy terminus of the beta ENaC. CONCLUSIONS: This new mutation leading to Liddle's syndrome highlights the importance of the carboxy terminus of the beta ENaC in the activity of the epithelial sodium channel. Small doses of amiloride are able to control the blood pressure on a long-term basis in this monogenic form of hypertension.

Properties of KvLQT1 K+ channel mutations in Romano-Ward and Jervell and Lange-Nielsen inherited cardiac arrhythmias.

Mutations in the delayed rectifier K+ channel subunit KvLQT1 have been identified as responsible for both Romano-Ward (RW) and Jervell and Lange-Nielsen (JLN) inherited long QT syndromes. We report the molecular cloning of a human KvLQT1 isoform that is expressed in several human tissues including heart. Expression studies revealed that the association of KvLQT1 with another subunit, IsK, reconstitutes a channel responsible for the IKs current involved in ventricular myocyte repolarization. Six RW and two JLN mutated KvLQT1 subunits were produced and co-expressed with IsK in COS cells. All the mutants, except R555C, fail to produce functional homomeric channels and reduce the K+ current when co-expressed with the wild-type subunit. Thus, in both syndromes, the main effect of the mutations is a dominant-negative suppression of KvLQT1 function. The JLN mutations have a smaller dominant-negative effect, in agreement with the fact that the disease is recessive. The R555C subunit forms a functional channel when expressed with IsK, but with altered gating properties. The voltage dependence of the activation is strongly shifted to more positive values, and deactivation kinetics are accelerated. This finding indicates the functional importance of a small positively charged cytoplasmic region of the KvLQT structure where two RW and one JLN mutations have been found to take place.

New modulatory alpha subunits for mammalian Shab K+ channels.

Two novel K+ channel alpha subunits, named Kv9.1 and Kv9.2, have been cloned. The Kv9.2 gene is situated in the 8q22 region of the chromosome. mRNAs for these two subunits are highly and selectively expressed in the nervous system. High levels of expressions are found in the olfactory bulb, cerebral cortex, hippocampal formation, habenula, basolateral amygdaloid nuclei, and cerebellum. Interestingly Kv9.1 and Kv9.2 colocalized with Kv2.1 and/or Kv2.2 alpha subunits in several regions of the brain. Neither Kv9.1 nor Kv9.2 have K+ channel activity by themselves, but both modulate the activity of Kv2.1 and Kv2.2 channels by changing kinetics and levels of expression and by shifting the half-inactivation potential to more polarized values. This report also analyzes the changes in electrophysiological properties of Kv2 subunits induced by Kv5.1 and Kv6.1, two other modulatory subunits. Each modulatory subunit has its own specific properties of regulation of the functional Kv2 subunits, and they can lead to extensive inhibitions, to large changes in kinetics, and/or to large shifts in the voltage dependencies of the inactivation process. The increasing number of modulatory subunits for Kv2.1 and Kv2.2 provides an amazingly new capacity of functional diversity.

The structure, function and distribution of the mouse TWIK-1 K+ channel.

The two P domain K+ channel mTWIK-1 has been cloned from mouse brain. In Xenopus oocytes, mTWIK-1 currents are K+-selective, instantaneous, and weakly inward rectifying. These currents are blocked by Ba2+ and quinine, decreased by protein kinase C and increased by internal acidification. The apparent molecular weight of mTWIK-1 in brain is 81 kDa. A 40 kDa form is revealed after treatment with a reducing agent, strongly suggesting that native mTWIK-1 subunits dimerize via a disulfide bridge. TWIK-1 mRNA is expressed abundantly in brain and at lower levels in lung, kidney, and skeletal muscle. In situ hybridization shows that mTWIK-1 expression is restricted to a few brain regions, with the highest levels in cerebellar granule cells, brainstem, hippocampus and cerebral cortex.

Cloning, functional expression and brain localization of a novel unconventional outward rectifier K+ channel.

Human TWIK-1, which has been cloned recently, is a new structural type of weak inward rectifier K+ channel. Here we report the structural and functional properties of TREK-1, a mammalian TWIK-1-related K+ channel. Despite a low amino acid identity between TWIK-1 and TREK-1 (approximately 28%), both channel proteins share the same overall structural arrangement consisting of two pore-forming domains and four transmembrane segments (TMS). This structural similarity does not give rise to a functional analogy. K+ currents generated by TWIK-1 are inwardly rectifying while K+ currents generated by TREK-1 are outwardly rectifying. These channels have a conductance of 14 pS. TREK-1 currents are insensitive to pharmacological agents that block TWIK-1 activity such as quinine and quinidine. Extensive inhibitions of TREK-1 activity are observed after activation of protein kinases A and C. TREK-1 currents are sensitive to extracellular K+ and Na+. TREK-1 mRNA is expressed in most tissues and is particularly abundant in the lung and in the brain. Its localization in this latter tissue has been studied by in situ hybridization. TREK-1 expression is high in the olfactory bulb, hippocampus and cerebellum. These results provide the first evidence for the existence of a K+ channel family with four TMS and two pore domains in the nervous system of mammals. They also show that different members in this structural family can have totally different functional properties.

K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current.

In mammalian cardiac cells, a variety of transient or sustained K+ currents contribute to the repolarization of action potentials. There are two main components of the delayed-rectifier sustained K+ current, I(Kr) (rapid) and I(Ks), (slow). I(Kr) is the product of the gene HERG, which is altered in the long-QT syndrome, LQT2. A channel with properties similar to those of the I(Ks) channel is produced when the cardiac protein IsK is expressed in Xenopus oocytes. However, it is a small protein with a very unusual structure for a cation channel. The LQT1 gene is another gene associated with the LQT syndrome, a disorder that causes sudden death from ventricular arrhythmias. Here we report the cloning of the full-length mouse K(V)LQT1 complementary DNA and show that K(V)LQT1 associates with IsK to form the channel underlying the I(Ks) cardiac current, which is a target of class-III anti-arrhythmic drugs and is involved in the LQT1 syndrome.

A new K+ channel beta subunit to specifically enhance Kv2.2 (CDRK) expression.

Cloned K+ channel beta subunits are hydrophilic proteins which associate to pore-forming alpha subunits of the Shaker subfamily. The resulting alphabeta heteromultimers K+ channels have inactivation kinetics significantly more rapid than those of the corresponding alpha homomultimers. This paper reports the cloning and the brain localization of mKvbeta4 (m for mouse), a new beta subunit. This new beta subunit is highly expressed in the nervous system but is also present in other tissues such as kidney. In contrast with other beta subunits, coexpression of the mKvbeta4 subunit with alpha subunits of Shaker-type K+ channel does not modify the kinetic properties or voltage-dependence of these channels in Xenopus oocytes. Instead, mKvbeta4 associates to Kv2.2 (CDRK), a Shab K+ channel, to specifically enhance (a factor of up to 6) its expression level without changing its elementary conductance or kinetics. It is without effect on another closely related Shab K+ channel Kv2.1 (DRK1). Chimeras between Kv2.1 and Kv2. 2 indicate that the COOH-terminal end of the Kv2.2 protein is essential for its mKvbeta4 sensitivity. The functional results associated with the observation of the co-localization of mKvbeta4 and Kv2.2 transcripts in most brain areas strongly suggest that both subunits interact in vivo to form a slowly-inactivating K+ channel. A chaperone-like effect of mKvbeta4 seems to permit the integration of a larger number of Kv2.2 channels at the plasma membrane.

Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties towards Shab and Shaw channels.

Outward rectifier K+ channels have a characteristic structure with six transmembrane segments and one pore region. A new member of this family of transmembrane proteins has been cloned and called Kv8.1. Kv8.1 is essentially present in the brain where it is located mainly in layers II, IV and VI of the cerebral cortex, in hippocampus, in CA1-CA4 pyramidal cell layer as well in granule cells of the dentate gyrus, in the granule cell layer and in the Purkinje cell layer of the cerebellum. The Kv8.1 gene is in the 8q22.3-8q24.1 region of the human genome. Although Kv8.1 has the hallmarks of functional subunits of outward rectifier K+ channels, injection of its cRNA in Xenopus oocytes does not produce K+ currents. However Kv8.1 abolishes the functional expression of members of the Kv2 and Kv3 subfamilies, suggesting that the functional role of Kv8.1 might be to inhibit the function of a particular class of outward rectifier K+ channel types. Immunoprecipitation studies have demonstrated that inhibition occurs by formation of heteropolymeric channels, and results obtained with Kv8.1 chimeras have indicated that association of Kv8.1 with other types of subunits is via its N-terminal domain.