Today’s post links together subjects that have been covered previously.
It does suggest that there are multiple therapies that may be effective in the large sub-group of autism that is characterized by the neurotransmitter GABA being excitatory (E) rather than inhibitory (I). The science was covered in the earlier very complicated post:-
The growing list of potential therapies is:-
· Bumetanide (awaiting funding for Stage 3 clinical trials in humans)
· Micro-dose Clonazepam (trials in mouse models of autism)
· Diamox (off-label use in autism)
· Potassium Bromide - to be covered in a later post (in use for 150 years)
Not surprisingly, all of these drugs also have an effect on certain types of seizure.
The optimal therapy in people with this E/I imbalance will likely be a combination of some of the above.
Periodic paralysis (Hypokalemic periodic paralysis or HypoPP) is a rare condition that causes temporary paralysis that can be reversed by taking potassium. A similar condition is hypokalemic sensory overload, when someone becomes overwhelmed by lights or sounds, but after taking potassium all goes back to normal. Autistic sensory overload, experienced by most people with autism, can also be reduced by potassium.
Though rare, we know that HypoPP is caused by dysfunction in the ion channels Nav1.4 and/or Cav1.1.
For decades one of the treatments for HypoPP has been a diuretic called Diamox/Acetazolamide.
Other treatments include raising potassium levels using supplements or potassium sparing diuretics.
Bumetanide is a diuretic, but rather than raising potassium levels, it does the opposite. So I always thought it was odd that bumetanide would have a positive effect on HypoPP. But the research showed a benefit.
Autism and Channelopathies
We know that autism and epilepsy are associated with various ion channel and transporter dysfunctions (channelopathies). In a recent post I was talking about Cav1.1 to Cav1.4.
Today we are talking about Cav1.1 and Nav1.4.
We know that Nav1.1 is associated with epilepsy and some autism (Dravet syndrome).
Nav1.4 is expressed at high levels in adult skeletal muscle, at low levels in neonatal skeletal muscle, and not at all in brain
Nav1.1 expression increases during the third postnatal week and peaks at the end of the first postnatal month, after which levels decrease by about 50% in the adult.
We saw with calcium channels that a dysfunction in one of Cav1.1 to Cav1.4 can cause a dysfunction in another dysfunction in another one of Cav1.1 to Cav1.4.
We also so that in autism the change in expression of NKCC1 and KCC2 as the brain matures failed to occur and so in effect they remain immature and therefore malfunction.
So it is plausible that sodium channels may also malfunction in a similar way.
Hypokalemic periodic paralysis (hypoPP) is an autosomal dominant neuromuscular disorder characterized by episodes of flaccid skeletal muscle paralysis accompanied by reduced serum potassium levels. It is caused by mutations in one of two sarcolemmal ion channel genes, CACNA1S and SCN4A1-3 that lead to dysfunction of the dihydropyridine receptor or the alpha sub-unit of the skeletal muscle voltage gated sodium channel Nav1.4. Seventy to eighty percent of cases are caused by mutations of CACNA1S and ten percent by mutations of SCN4A4.
There are no consensus guidelines for the treatment of hypoPP. Current pharmacological agents commonly used include potassium supplements, potassium sparing diuretics and carbonic anhydrase inhibitors (acetazolamide and dichlorphenamide). Dichlorphenamide is the only therapy for hypoPP to have undergone a randomized double blind placebo controlled cross over trial. This trial showed a significant efficacy of dichlorphenamide in reducing attack frequency but the inclusion criteria were based on clinical diagnosis of hypoPP and not genetic confirmation.
Cav1.1 also known as the calcium channel, voltage-dependent, L type, alpha 1S subunit, (CACNA1S), is a protein which in humans is encoded by the CACNA1S gene
The Nav1.4 voltage-gated sodium channel is encoded by the SCN4A gene. Mutations in the gene are associated with hypokalemic periodic paralysis, hyperkalemic periodic paralysis, paramyotonia congenita, and potassium-aggravated myotonia.
Ranolazine is an antianginal and anti-ischemic drug that is used in patients with chronic angina. Ranzoline blocks Na+ currents of Nav1.4. Both muscle and neuronal Na+ channels are as sensitive to ranolazine block as their cardiac counterparts. At its therapeutic plasma concentrations, ranolazine interacts predominantly with the open but not resting or inactivated Na+ channels. Ranolazine block of open Na+ channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate.
Muscle channelopathies:does the predicted channel gating pore offer new treatment insights for hypokalaemic periodic paralysis?
Transient attacks of weakness in hypokalaemic periodic paralysis are caused by reduced fibre excitability from paradoxical depolarization of the resting potential in low potassium. Mutations of calcium channel and sodium channel genes have been identified as the underlying molecular defects that cause instability of the resting potential. Despite these scientific advances, therapeutic options remain limited. In a mouse model of hypokalaemic periodic paralysis from a sodium channel mutation (NaV1.4-R669H), we recently showed that inhibition of chloride influx with bumetanide reduced the susceptibility to attacks of weakness, The R528H mutation in the calcium channel gene ( . encoding CaV1.1) is the most common cause of hypokalaemic periodic paralysis. We developed a CaV1.1-R528H knock-in mouse model of hypokalaemic periodic paralysis and show herein that bumetanide protects against both muscle weakness from low K+ challenge and loss of muscle excitability from a glucose plus insulin infusion. This work demonstrates the critical role of the chloride gradient in modulating the susceptibility to ictal weakness and establishes bumetanide as a potential therapy for hypokalaemic periodic paralysis arising from either NaV1.4 or CaV1.1 mutations.
Mode of action
The research does state that nobody knows why Diamox is effective in many cases of hypoPP.
My reading of the research has already taken me in a different direction. While researching the GABAA receptor that is dysfunctional in some autism, it occurred to me that in addition to targeting the NKCC1 receptor with bumetanide, another way of lowering chloride levels within the cells might well exist.
I suggested in an earlier post that Diamox could be used to target the AE3 exchanger.
What Diamox (acetazolamide) does is lower the pH of the blood in the following way.
Acetazolamide is a carbonic anhydrase inhibitor, hence causing the accumulation of carbonic acid Carbonic anhydrase is an enzyme found in red blood cells that catalyses the following reaction:
hence lowering blood pH, by means of the following reaction that carbonic acid undergoes
In doing so there will be an effect on both AE3 and NDAE, below. This will change the intracellular concentration of Cl-, and hence give a similar result to bumetanide.
This would also explain the phenomenon cited below that pH affects the excitability of the brain.
Over excitability of the brain is the cause of some of the effects seen as autism and clearly Over excitability of the brain will be the cause of some people’s seizures/epilepsy.
Not surprisingly, then one of the uses of Diamox is to avoid seizures.
Anion exchanger 3 (AE3) in autism
Anion exchange protein 3 is a membrane transport protein that in humans is encoded by the SLC4A3 gene. It exchanges chloride for bicarbonate ions. It increases chloride concentration within the cell. AE3 is an anion exchanger that is primarily expressed in the brain and heart
Its activity is sensitive to pH. AE3 mutations have been linked to seizures
Bicarbonate (HCO3-) transport mechanisms are the principal regulators of pH in animal cells. Such transport also plays a vital role in acid-base movements in the stomach, pancreas, intestine, kidney, reproductive organs and the central nervous system.
Two types of chloride transporters are required for GABAA receptor-mediated inhibition in C. elegans
Chloride influx through GABA-gated Cl− channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl− gradient established in part by K+–Cl− cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na+-driven Cl−–HCO3− exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K+–Cl− cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl− extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl− gradient such that Cl− flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl− gradient. Our results show that Na+-driven Cl−–HCO3− exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.
During early development, γ-aminobutyric acid (GABA) depolarizes and excites neurons, contrary to its typical function in the mature nervous system. As a result, developing networks are hyperexcitable and experience a spontaneous network activity that is important for several aspects of development. GABA is depolarizing because chloride is accumulated beyond its passive distribution in these developing cells. Identifying all of the transporters that accumulate chloride in immature neurons has been elusive and it is unknown whether chloride levels are different at synaptic and extrasynaptic locations. We have therefore assessed intracellular chloride levels specifically at synaptic locations in embryonic motoneurons by measuring the GABAergic reversal potential (EGABA) for GABAA miniature postsynaptic currents. When whole cell patch solutions contained 17–52 mM chloride, we found that synaptic EGABA was around −30 mV. Because of the low HCO3− permeability of the GABAA receptor, this value of EGABA corresponds to approximately 50 mM intracellular chloride. It is likely that synaptic chloride is maintained at levels higher than the patch solution by chloride accumulators. We show that the Na+-K+-2Cl− cotransporter, NKCC1, is clearly involved in the accumulation of chloride in motoneurons because blocking this transporter hyperpolarized EGABA and reduced nerve potentials evoked by local application of a GABAA agonist. However, chloride accumulation following NKCC1 block was still clearly present. We find physiological evidence of chloride accumulation that is dependent on HCO3− and sensitive to an anion exchanger blocker. These results suggest that the anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons.
So the science does confirm that “chloride accumulation following NKCC1 block was still clearly present”. This means that bumetanide is likely only a partial solution.
We also see that “anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons” and “that chloride accumulation that is dependent on HCO3−”.
This is a subject of some research, but it is still early days.
Bicarbonate homeostasis in excitable tissues: role of AE3 Cl−/HCO3−exchanger and carbonic anhydrase XIV interaction
I suggest that Diamox, via its effect on HCO3−, may affect anion exchanger AE3 and further reduce chloride accumulation within cells. This may have a further cumulative effect on GABA.
As we saw earlier, bumetanide does indeed shift GABA from excitatory to inhibitory in people who neurons remain in an immature state (like those of a typical two week old baby). To my surprise, the use of micro-dose Clonazepam, as proposed by Professor Catterall, but in addition to Bumetanide, has a further effect on GABA’s excitatory/inhibitory imbalance.
Taken together this would highlight the possible further benefit of Diamox.
Normal blood pH is tightly regulated between 7.35 and 7.45. I do wonder if perhaps in some people with autism, the pH of their blood is slightly elevated (alkaline), this would contribute to excitability of the brain.
Since Diamox increases the oxygen carrying capacity of the blood, I further wonder if this additional oxygen may also be beneficial in some cases. Since some people are adamant that hypobaric oxygen therapy has beneficial (although not sustained) effects in autism, surely a better treatment would be Diamox?
Since the body is controlled via so-called feedback loops, perhaps in a small subset of people with autism who respond to extra O2, they actually have blood pH that is higher than 7.45. In which case measuring blood pH would be a biomarker of who would respond to hypobaric oxygen therapy. Not surprisingly then, trials of hypobaric oxygen therapy in autism fail, because most of the trial subjects do not have elevated blood pH.
So there are many reasons that Diamox should be trialed in autism. I did find one (DAN) doctor currently using it, but they do not really explain why.