Retigabine: Bending Potassium Channels to Our Will

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1201633/

The New Anticonvulsant Retigabine Favors Voltage-dependent Opening of the Kv7.2 (KCNQ2) Channel by Binding to Its Activation Gate
Wuttke TV, Seebohm G, Bail S, Maljevic S, Lerche H

Mol Pharmacol 2005;67:1009–1017 [PubMed]

Retigabine (RTG) is an anticonvulsant drug with a novel mechanism of action. It activates neuronal KCNQ-type K+ channels by inducing a large hyperpolarizing shift of steady-state activation. To identify the structural determinants of KCNQ channel activation by RTG, we constructed a set of chimeras by using the neuronal KV7.2 (KCNQ2) channel, which is activated by RTG, and the cardiac KV7.1 (KCNQ1) channel, which is not affected by this drug. Substitution of either the S5 or the S6 segment in KV7.2 by the respective parts of KV7.1 led to a complete loss of activation by RTG. Trp236 in the cytoplasmic part of S5 and the conserved Gly301 in S6 (KV7.2), considered as the gating hinge (Ala336 in KV7.1), were found to be crucial for the RTG effect: mutation of these residues could either knock out the effect in KV7.2 or restore it partially in KV7.1/KV7.2 chimeras. We propose that RTG binds to a hydrophobic pocket formed upon channel opening between the cytoplasmic parts of S5 and S6 involving Trp236 and the channel's gate, which could well explain the strong shift in voltage-dependent activation.

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COMMENTARY
Voltage-gated ion channels are important determinants of neuronal activity. Numerous ion-channel mutations have been linked to epilepsy, and many antiepileptic medications modulate sodium or calcium channels. Potassium channels are probably as important in regulating membrane potential, yet little has been done to develop therapeutic modulators of this protein. The article by Wuttke et al. explores the mechanism whereby a novel antiepileptic medication, retigabine (RTG), enhances a subset of voltage-gated potassium channels.

The canonical structure for voltage-gated ion channels is a combination of four cassettes, each made of six transmembrane (TM) domains. Whereas this structure is typical of sodium and calcium channels, which have all four cassettes contained within a single large protein, the arrangement is much more complicated for potassium channels. More than 80 human potassium channel–related genes have been identified, many with multiple splice variants and accessory proteins (1). This diversity allows a wide range of channels, some of which are voltage sensitive, whereas others respond to many different stimuli, including calcium, G proteins, ATP, and changes in pH. Fortunately, the multitude of potassium channels all are based on a common structural motif, two TM domains (M1 and M2) and an intervening P loop. Voltage-gated K+channels (Kv) contain six TM domains, with TM1–4 serving as the voltage sensor (2) and TM5–6 being analogous to M1-P–M2 in other K+ channel families.

A combination of four of these motifs is required to form functional potassium channels. A key feature of potassium channels is their ability to allow the selective passage of K+ but not of other cations. A pore is formed at the interface of the four TM6 (M2 in non-Kv channels) segments. However, the selectivity filter also includes a section of the extracellular P loop, which folds back down and lines the extracellular portion of the pore. Beyond the selectivity filter, closer to the intracellular membrane, the four TM6 domains of a resting potassium channel are closely juxtaposed, forming a barrier to ion flow. This region, known as the gate, is the putative site of retigabine action. With channel activation, TM6 is thought to bend at a “hinge” near its midsection, which contains a relatively conserved glycine. As depicted in Figure 1, this event allows the intracellular portion of the pore to widen, permitting passage of ions (3).


FIGURE 1
Model of potassium channel gating. A: The TM6 domain of KV is relatively linear at rest. Activation involves a swivel and bending motion proximate to a “hinge” region near the middle of TM6. B: In the intact channel, four linear TM6 domains ...
RTG is an antiepileptic medication undergoing clinical trials. It is effective for a wide range of seizure models and has few drug interactions (4). RTG has several pharmacologic actions, but an interesting and unique action is its ability to enhance KCNQ2/KCNQ3 potassium channels (which under the new nomenclature also are designated KV7.2 and KV7.3). KCNQ2/KCNQ3 heterotetramers form the voltage-gated ion channel that mediates the M-current, which regulates the subthreshold excitability and responsiveness to neurotransmitters of many neurons. Interestingly, mutations in the genes encoding KCNQ2 and KCNQ3 are associated with the epilepsy syndrome, benign familial neonatal convulsions. The M-current is somewhat active at resting membrane potential and then slowly increases activity during depolarization. Its slow kinetics probably preclude an involvement in shaping individual action potentials but allow the M-current to alter spike frequency during bursts of neuronal firing. RTG affects the M-current in multiple ways, including accelerating activation, slowing deactivation, and shifting the voltage activation curve by 30 mV toward more hyperpolarized potentials. How RTG causes these changes is the focus of the present article.

To help elucidate the mechanism of potassium channel modulation, the authors took advantage of the fact that whereas KCNQ2-5 respond to RTG, the closely related cardiac channel KCNQ1 does not. Chimeric proteins were generated, and segments of KCNQ1were transplant into KCNQ2 proteins and vice versa. Electrophysiological recordings from oocytes expressing these recombinant proteins demonstrated that potassium-current enhancement by RTG involves TM5 and TM6. The TM5 and TM6 regions of the KCNQ1 and KCNQ2 proteins differ by only 13 amino acids. KCNQ2 channels lost RTG sensitivity when a single tryptophan in TM5 was mutated to leucine, as found in KCNQ1 proteins. To investigate the role of TM6 in RTG action, the authors made use of the fact that KCNQ1 is one of the few potassium channels without glycine in the putative hinge segment. As previously mentioned, this site is important in channel opening; thus, mutating the glycine in most potassium channels results in a nonfunctional protein. How KCNQ1 proteins can function without the glycine is unknown, but the phenomenon may suggest some differences in channel activation for this particular Kv channel. Nonetheless, as might be expected, mutating glycine in KCNQ2 to alanine resulted in a channel that could not open. Conversely, a KCNQ2 protein containing the KCNQ1 TM6 sequence produces a channel that is functional but insensitive to RTG. However, when that single glycine is reintroduced into the homologous position of TM6, the channel regains RTG sensitivity. The authors propose a model in which RTG binds in a pocket between TM5 and TM6, thus stabilizing the bend in TM6 that is thought to occur on opening. This event would theoretically reduce the energy required for activation (so less depolarization is needed to open the channel) and explain the faster activation and slower deactivation induced by RTG. The authors' theory fits well with single-channel data that RTG prolongs both open states by two- to fourfold (5). A more recent article by Schenzer et al. (6) found similar structural requirements for RTG sensitivity by using Kv7.3 proteins.

Chimera and point mutation studies come with several important caveats. First, although these techniques can help identify residues that are important in RTG action, it is often difficult to tell whether the sites involve binding, transduction, or some other process distant from the binding site. This issue is made particularly evident by the number of nonfunctional chimeras and mutants the authors screened. Use of this screening technique does not make it possible to assess whether the mutants would have retained RTG sensitivity if they had been able to open at all. The authors did not determine whether the RTG-insensitive mutants had lost RTG binding or the ability of RTG to alter gating. It is likely that none of these residues acts alone; the process of activation probably involves many interactions among many residues. To fully understand residue functions, mutating several points at once—to either abolish or restore drug effect—is often required (7). Despite the limitations of the studies by Wuttke and colleagues, they provide important clues about the mechanism for a potentially unique and effective tool to modulate potassium channels in epilepsy.

 

Soo, could you analyze the article for us if you found something new? Study is 10 years old and I didn't find anything new on it - atleast not regarding tinnitus?

 

It's just an insight on how trobalt functions. It's important to note how the drug works/functions.

 

"The pharmacologic profile of retigabine [RTG (international nonproprietary name); ezogabine, EZG (U.S. adopted name)], is different from all currently approved antiepileptic drugs (AEDs). Its primary mechanism of action (MoA) as a positive allosteric modulator of KCNQ2–5 (Kv7.2–7.5) ion channels defines RTG/EZG as the first neuronal potassium (K+ ) channel opener for the treatment of epilepsy. KCNQ2–5 channels are predominantly expressed in neurons and are important determinants of cellular excitability, as indicated by the occurrence of human genetic mutations in KCNQ channels that underlie inheritable disorders including, in the case of KCNQ2/3, the syndrome of benign familial neonatal convulsions. In vitro pharmacologic studies demonstrate that the most potent action of RTG/EZG is at KCNQ2–5 channels, particularly heteromeric KCNQ2/3. Furthermore, mutagenesis and modeling studies have pinpointed the RTG/EZG binding site to a hydrophobic pocket near the channel gate, indicating how RTG/EZG can stabilize the open form of KCNQ2–5 channels; the absence of this site in KCNQ1 also provides a clear explanation for the inbuilt selectivity RTG/EZG has for potassium channels other than the KCNQ cardiac channel. KCNQ channels are active at the normal cell resting membrane potential (RMP) and contribute a continual hyperpolarizing influence that stabilizes cellular excitability. The MoA of RTG/EZG increases the number of KCNQ channels that are open at rest and also primes the cell to retort with a larger, more rapid, and more prolonged response to membrane depolarization or increased neuronal excitability. In this way, RTG/EZG amplifies this natural inhibitory force in the brain, acting like a brake to prevent the high levels of neuronal action potential burst firing (epileptiform activity) that may accompany sustained depolarizations associated with the initiation and propagation of seizures. This action to restore physiologic levels of neuronal activity is thought to underlie the efficacy of RTG/EZG as an anticonvulsant in a broad spectrum of preclinical seizure models and in placebo controlled trials in patients with partial epilepsy. In this article, we consider the pharmacologic characteristics of RTG/EZG at the receptor, cellular, and network levels as a means of understanding the novel and efficacious MoA of this new AED as defined in both preclinical and clinical research."

 

I need to be a neuroscientist to understand this lot.

 

http://www.ncbi.nlm.nih.gov/pubmed/22220513

The mechanism of action of retigabine (ezogabine), a first-in-class K+ channel opener for the treatment of epilepsy.
Gunthorpe MJ1, Large CH, Sankar R.
Author information

Abstract
The pharmacologic profile of retigabine [RTG (international nonproprietary name); ezogabine, EZG (U.S. adopted name)], is different from all currently approved antiepileptic drugs (AEDs). Its primary mechanism of action (MoA) as a positive allosteric modulator of KCNQ2-5 (K(v) 7.2-7.5) ion channels defines RTG/EZG as the first neuronal potassium (K(+)) channel opener for the treatment of epilepsy. KCNQ2-5 channels are predominantly expressed in neurons and are important determinants of cellular excitability, as indicated by the occurrence of human genetic mutations in KCNQ channels that underlie inheritable disorders including, in the case of KCNQ2/3, the syndrome of benign familial neonatal convulsions. In vitro pharmacologic studies demonstrate that the most potent action of RTG/EZG is at KCNQ2-5 channels, particularly heteromeric KCNQ2/3. Furthermore, mutagenesis and modeling studies have pinpointed the RTG/EZG binding site to a hydrophobic pocket near the channel gate, indicating how RTG/EZG can stabilize the open form of KCNQ2-5 channels; the absence of this site in KCNQ1 also provides a clear explanation for the inbuilt selectivity RTG/EZG has for potassium channels other than the KCNQ cardiac channel. KCNQ channels are active at the normal cell resting membrane potential (RMP) and contribute a continual hyperpolarizing influence that stabilizes cellular excitability. The MoA of RTG/EZG increases the number of KCNQ channels that are open at rest and also primes the cell to retort with a larger, more rapid, and more prolonged response to membrane depolarization or increased neuronal excitability. In this way, RTG/EZG amplifies this natural inhibitory force in the brain, acting like a brake to prevent the high levels of neuronal action potential burst firing (epileptiform activity) that may accompany sustained depolarizations associated with the initiation and propagation of seizures. This action to restore physiologic levels of neuronal activity is thought to underlie the efficacy of RTG/EZG as an anticonvulsant in a broad spectrum of preclinical seizure models and in placebo-controlled trials in patients with partial epilepsy. In this article, we consider the pharmacologic characteristics of RTG/EZG at the receptor, cellular, and network levels as a means of understanding the novel and efficacious MoA of this new AED as defined in both preclinical and clinical research.

 

That's it's useless for a normal user of the forum we aren't scentist or neuroscentist. In this forum there are only normal people with a target. Eliminate tinnitus or finding a relief. I'm a electricist and I know how function a resistence but I don't know how fucntion at fisic level that's is useless!!

 

If you step through the papers word by word and search the web, you may be surprised how much you can learn about these topics in a short amount of time. I really appreciate the postings Danny Boy.

 

Dr daniel

 

@Jokko this is what you need to know tinnitus since 03-2015. Currently at 200 mg three times a day, last pill at 4 am, its 10 am know and i can barely hear it (1). I have been taking it for a month, left it for a week aprox. because stock run out, had a few shaky days (neurons started overacting again, left the pills cold turkey) but three days later it lower itself to a 3 (even in silence), i would had been very happy at this level. Got pills back and back on the horse again taking 200 mg three times a day trying to get rid of it all together. Your tinnitus started in 01.2015 i think you will benefit from it if you can get it.

 

@papu I understand you but you know my tinnitus isn't so bad to take a risiko drug like trobalt. Trobalt have some collateral effect that can be very devastating and permanent. Also now I take antypsychotic drug and SSRI drug that are psycodrug if I take trobalt maybe the side effect are more potent like if you don't take anything

 

@Jokko I agree with you it has to be a very personal decision, its true what you say, side effects can be very harmful for this pill. if your at peace with your condition its not worth it. Hopefully sf 00034 or aut63 will be better for you.


on a personal note I did have a terrible overactive bladder *had to go to the rest room to pee at least three times in a 2 hour movie*, now that is gone !!!!

 

I'm not an expert, but it can be resumed in few words. Everybody knows what a cell is, that is, a small container of proteins and other molecules inside an envelope, the cell membrane. This membrane is made of phospholipids, a special kind of fat that is almost free-flowing but prevents ions to go inside or outside the cell. It's a tiny (7 nm width) envolture and it's water-proof. Most proteins are placed in this membrane and allow for a variety of functions: transport of nutrients, of proteins, production of energy, or to maintain ion homeostasis.

Most cells in the body have small electric activity, but in the brain, neurons are specialized in this kind of activity. And, how a cell is able to transmit electricity or electric signals? Usually, there is an imbalance of the ions inside and outside the cell membrane. The part inside the cell is called cytoplasm, while the outside part is called the extracellular space. Ions are small, charged atoms. For example, Na+ (sodium), Ca2+ (calcium), K+ (our loved potassium) or Cl- (chlorine). So, an imbalance of charged particles lead to the development of an electrical field. The strength of this field is small, and a cell in rest has a transmembrane potential of around -40 mV. When a neuron starts signaling, there is an entering of Na+ ions into the cell membrane, and the potential goes from -40 mV to +100 mV approx. The neuron is "active" and "depolarized". Then, the neuron needs to dissipate this potential to enter the "rest state" again, and does this by leaking out K+ ions and returning to the rest state, polarizing again the membrane. Who does this? The K+ voltage-gated ion channels, the famous Kv channels. Activated by the voltage, they open and let K+ go out to the extracellular space.

When the potential is restored, they close and the neuron goes back to resting state, and stops "firing". This is an overly simplified explanation but IMHO is enough to understand what's going on. In our case, as tinnitus or epilepsy develops, those channels get stuck in the "closed" mode, no matter the voltage, and this is where drugs as retigabine or AUT (a hydantoin derivative) interact with the potassium channels opening them, in a reversible fashion, letting K+ ions out and hyperpolarizing again the cell to its -40 mV rest state, stopping firing and calming its electric activity. Again, this is a simplified view.

If all the research done in this field is true, and I don't see the reason it is not (papers get a long and strict revision process to get published, if a mistake is done it will get caught, plus usually several research teams are working simultaneously in the same topic and if some of them does not arrive to the same conclusions, something is going wrong), tinnitus is nothing more than a group of neurons stuck in the polarized or active mode and they only need a way to get back to the resting state. Again, things are more complicated than that. I mean, neuronal plasticity, but I'm not a proper neuroscientist and could not explain that clearly.

At the end, you need to reverse this state and for now, retigabine acts this way and it looks like it's working, besides the side effects. Autifony's drug has the same mechanism of action, so it seems plausible that will work also. Also with the new SF34 (RTG on steroids) or whatever molecule will come up in the next few years.

In fact, if I had enough money, I would immediately start growing cultures with Kv3.x overexpression and screening lots of compounds, to see what opens the channels and then go directly to animal testing and then clinical trials, but I have not the money or the resources. Autifony are the pioneers and probably will get all the money. The same happened with benzos, once the first compound came out, tens of derivatives came later, with different potency, specificity, half-life and pharmacokinetics, so today an anxiety sufferer has lots of options to choose from.

Sorry for the long post, it's only my opinion. I could be wrong, but things are too logical from this point of view.

 

Nice explanation of brain state occurred by t.

In your point of view where do u see glutamate? As glutamate is stated to be main potassium channel blocker. Does opening K channels mean- removing glutamate by some kind of chemistry action (done by drug).

tnx

 

Again, I'm not working on neuro, but what glutamate does is to facilitate the entering of cations inside the cell in the axon, that is, Ca2+ and Na+, thus helping in the depolarization, or "activating" the cell. Transmembrane potential goes up, cell activates, starts firing and then all the nasty effects. It's known that lidocaine, a calcium channel blocker, suppresses tinnitus in most patients, but by a "shotgun" approach, wider than retigabine even.

 

I had to , mine was so bad and it insresed itself 6 times so , i did nto had a choice, when you get really bad u will take it.