Summary of Presentations Neurosciences Fundamentals 2017
FIRST YEAR MEDICAL STUDENTS UNIVERSIDAD CENTRAL DEL CARIBE | Neuroscience | March 2017 |
Presentations 2017, Medical Students First year. UCC
Resting Membrane Potential Gabriel Colón; Jorge Irizarry; Daphne Jorge Bezares; Andrea Medina; Adrianna Mojica; Angélica Peña and Carlos Ramírez
Ions found in neurons There exists different concentrations of ions in the extracellular fluid versus the intracellular fluid, thus contributing to setting the resting membrane potential for the cell. Extracellularly there are ions of sodium, chloride, bicarbonate, calcium and potassium from greater to lesser quantities. See the table below for specific amounts of ions per liter in the extracellular (ECF) and intracellular fluid (ICF). Note that even though there is variability amongst the quantity of ions in the compartments, these must obey the principle of macroscopic electroneutrality, meaning that there must be the same concentration of Ion
ECF (mEq/L)
ICF (mEq/L)
Na+
140
14
K+
4
120
Ca 2+
2.5
1*10-4
Cl-
105
10
HCO3-
24
10
cations as of anions. (Constanzo, 2014)
How ions cross the membrane There are two ways molecules can be moved across a membrane, and the distinction has to do with whether or not energy is used. Passive mechanisms uses no energy, while active transport requires energy. Diffusion is the movement of particles down their gradient. In simple diffusion, ions move down their gradient through a membrane. A membrane transport channel helps facilitated diffusion along. Transport that directly uses ATP is primary active transport. Secondary active transport moves multiple molecules across the membrane, powering the uphill movement of one molecule(s) with the downhill movement of the other(s).
Equilibrium potential An equilibrium potential is described when the membrane potential strengthens enough so that the number of potassium ions entering the cell and leaving the cell is equal. This feature is accomplished with two driving forces, the electrical forces and diffusion forces, which must be balanced. Diffusion forces and the developed attraction force cause the electrical force across the membrane by ion charge. Electrical force drives potassium inside the cell. Equilibrium potential for any given ion may be calculated using the Nernst equation:
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Resting membrane potential and the role of Na/K ATPase The resting membrane potential is the potential difference that exists across a membrane of excitable cells at rest and it is established by diffusion potentials for various ions. Because the ions with the highest permeability at rest make the greatest contributions and the membrane is more permeable to K+ and Cl- than to Na+ and Ca2+, the resting membrane potential ranges from -70 to -80 mV. Moreover, the Na/K ATPase has two roles in creating the membrane resting potential: a small direct electrogenic contribution and a more important indirect contribution of maintaining the K+ concentration gradient.
Clinical correlations -POTASSIUM-AGGRAVATED MYOTONIA
-Potassium-aggravated myotonia is a disorder that affects skeletal muscles and prevents normal relaxation. It is usually perceivable beginning in childhood or adolescence. Myotonia causes muscle stiffness that worsens after exercise and eating potassiumrich foods such as bananas and potatoes. Unlike other forms of myotonia, this disease is not characterized by muscle weakness. Potassium-aggravated myotonia is caused by missense mutations in
SCN4A gene as it alters the structure and function of sodium channels causing inability to properly regulate ion flow, increasing the movement of sodium ions into skeletal muscle cells causing contractions. -CONGENITAL LONG QT SYNDROME
The congenital long QT syndrome (LQTS) is a rare condition of abnormal myocardial repolarization that is clinically characterized by an increased risk of potentially fatal ventricular arrhythmias (George, 2005). The syndrome also shows electrographic abnormalities, including prolongation of the QT interval and T wave abnormalities. The genetic basis of the disease include LQTS genes that encode cardiac ion channel subunits or proteins involved in modulating ionic currents. Mutations in these genes (like the KCNQ1 or SCN5A) cause the disease by prolonging the duration of the action potential (Crotti et.al, 2008).
GENERALIZED EPILEPSY WITH FEBRILE SEIZURES
Generalized epilepsy with febrile seizures affect 3% of all children under six years. Its symptoms usually start in a family member who had febrile convulsions symptoms include high temperatures. Patients could encounter Dravet syndrome or myoclonic-astatic epilepsy. It has been mapped a two putative loci in relation
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to such disorder in the SCN1A gene. We see a mutation in the voltagegated sodium channel beta1 subunit; this disrupts a disulfide bridge changing the immunoglobulin-like fold. This causes either an increase or decrease in sodium channel activity, which results in seizures (Wallece et. al, 1998).
References Cardozo, D. (2016). An intuitive approach to understanding the resting membrane potential. Advances in Physiology Education, 40(4), 543–547. https://doi.org/10.1152/advan.00049.20 16 Costanzo, L. S. (2014). Physiology (Fifth ed.). Philadelphia, PA: Elsevier. Potassium-aggravated myotonia. (2007). Genetics Home Reference. Retrieved March 21, 2017, from https://ghr.nlm.nih.gov/condition/potass ium-aggravated-myotonia#synonyms Wallace, R. H., Wang, D. W., Singh, R., Scheffer, I. E., George, A. L., Phillips, H. A., … Mulley, J. C. (1998). Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta1 subunit gene SCN1B. Nature Genetics, 19(4), 366–370. https://doi.org/10.1038/1252 George, A.L. (2005). Inherited disorders of voltage-gated sodium channels. Journal of Clinical Investigation, 115:1990-1999. Crotti, L., Celano, G., Dagradi, F., Schwartz, P.J. (2008). Congenital long QT syndrome. Orphanet Journal of Rare Diseases, 3:18
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Presentations 2017, Medical Students First year. UCC
Action Potentials & Saltatory Conduction Gabriel Díaz Pagán; Raymond Gerena; Isabel Guillén Méndez; Isabel Mayorga Pérez; Giancarlo Mignucci; Myrta Isabel Olivera and Carlos J. Pacheco
The Action Potential An efficient exchange between ions causes the development of a dynamic process known as the action potential, which is a phenomenon that occurs along nerve cells and serves as the basic mechanism for transmission of information in the nervous system. Action Potentials have three basic characteristics: all-or-none response, propagation, and stereotypical size and shape. In other words, action potentials either occur or do not occur. If so, the signal (current) is propagated along the axon until reach the nerve terminal.
Characteristics The normal action potential for a neuron has a period of brief depolarization followed by an extended period of repolarization. The resting membrane potential (RMP) is approximately -70 mV, due to almost all of the potassium (leak) channels being open. Although potassium move, no net movement occurs because the cell membrane is near potassium’s equilibrium potential. With all of this in mind, the RMP is at -70 mV because
the potassium conductance “permeability” is high, which drives the membrane potential near the equilibrium potential for potassium (90 mV). Chloride ion also has a high permeability, but the potassium ion plays a larger role. When a cell reaches threshold, an action potential occurs. During the upstroke of the action potential, the membrane is depolarized, which opens the activation gate. The activation gate remains open for a brief time, allowing Na+ to flow through the channel and into the cell. This depolarization causes the potential to approach sodium’s equilibrium potential (+65 mV). However, the membrane potential actually settles earlier due to the inactivation gate begin to act. Afterwards, the membrane potential repolarizes toward RMP, through the opening of potassium channels. In fact, the potassium conductance reaches a level higher than it was at rest, so the repolarization results in a membrane potential below RMP. Eventually, the potassium conductance returns to its resting level, and the membrane potential depolarizes slightly back to RMP. At this point, when Na+ Channels recovers from inactivation a new action potential can be generated.
The refractory period The refractory period determines whether a new action potential can be initiated. But, this action potential can be of the same magnitude or smaller
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during the relative refractory period. In the absolute refractory period, it is impossible to elicit a new action potential. This is because the inactivation gates of the Na+ channels is acting. On the other hand, the in the relative refractory period has the possibility to initiate a new action potential. This is because it appears right after the absolute refractory period and spans along the repolarization phase. During this time, the inward current necessary to develop an action potential has to be greater than normal because the K+ conductance is closer to its resting potential.
Autorregeneration Action potential at one site causes the regeneration of another action potential onto its adjacent site. It will be propagated by the spread of local currents down nerve’s axon, initiated by the voltage gradient between the active and inactive regions of the axon. This change is caused by reversing the polarity of the membrane potential, from negative to positive. The inward, positive sodium current flows causing adjacent inactive regions to depolarize to threshold. While continuous regions are being depolarized, the original active regions are being repolarized. This is caused by the loss of potassium ions, returning the membrane back to its resting potential (-70 mV).
Conduction Velocity Conduction velocity is the speed at which the action potential is propagated along the nerve fiber. Increasing the diameter of the nerve fiber lowers the resistance meaning that an action potential travels faster. Myelination of the nerve fiber helps increase membrane resistance and decrease membrane capacitance. Increased membrane resistance forces current to flow along the path of least resistance of the axon interior rather than across the high resistance path of the axonal membrane. Decreased membrane capacitance produces a decrease in time constant, at breaks in the myelin sheath, the axonal membrane depolarizes faster in response to inward current. Both of these effects result in increased conduction velocity.
Saltatory Conduction Although myelination increases the speed of action potentials, if the whole axon were covered by myelin, there would be no low resistance breaks in the membrane, therefore the depolarizing current would have nowhere to flow and this would mean no generation of action potentials. This myelin sheath is very important, especially in saltatory conduction. Myelinated areas do not have voltage gated ion channels, thus the movement of sodium and potassium ions is limited to the nodes of Ranvier. Instead of propagating via channels, the charge jumps from one node to the
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next. This process also helps save energy because the sodium-potassium pump will only be found in the node of Ranvier and will have less work to bring the ions back to resting levels, spending less ATP.
References: Constanzo, L. Physiology. 5th ed. Philadelphia, PA: Saunders/Elsevier, 2014. Pgs 19-24. Mougios, V. Exercise Biochemistry. Champaign, IL: Human Kinetics, 2006. Pg 93. Nolte, J. The Human Brain: An introduction to its functional anatomy. 6th ed. Philadelphia, PA: Mosby Elsevier, 2009. Pgs 165-167.
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Blockers of voltage activated ion channels Mónica Arias; Jorge Chéverez; Emanuel De Miranda; Carlos Guardia; Rolando J. Monteverde Díaz; Yiseiry Pérez Meléndez and Natalia Rodríguez Mañón
Introduction As we read through our neuroscience textbook, many processes are going on in our body to make this happen. From maintaining our heartbeat, to perceiving the different colors of our neuroanatomy atlas to distinguish the brain’s parietal lobe from the temporal lobe. These events require a fast communication between neurons, which happens through electrical messages achieved by the opening and closing of ion channels in cell membranes. Specifically, voltagegated ion channels are transmembrane proteins that open and close when activated and inactivated or deactivated, depending on changes at the electrical potential difference across the cell membrane. These voltage-gated channels are targets for many pharmacological agents, which modulate channel physiology and produce therapeutic, curative, palliative and/or adjuvant effects depending on the blocker of choice and what is being treated.
The Voltage gated Sodium Channel Voltage gated sodium channels are heteromeric transmembrane proteins. The alpha subunit is the selective sodium pore; it initiates voltagedependent activation after voltage changes. The beta subunit regulates alpha subunit properties. The sodium channel has three functional states: resting (closed, able to be active), open (activated), and inactivated (closed, non-ready to be active). Tetrodotoxin (TTX) is a pufferfish neurotoxin, which acts as a physical barrier, the neurotoxin positive charge interacts with the channel negative charge, blocking the sodium channel pore. Phenytoin is an antiseizure medication; it binds to the channel in its inactive state inhibiting depolarization of neurons in the motor cortex, preventing seizures spread.
Voltage gated Calcium Channels Calcium channels are members of a family of channels known as the voltage-gated channels. They play a key role for membrane depolarization in excitable cells. Two main families compose the calcium channel family: High Voltage-Activated (HVA) and Low Voltage-Activated (LVA) channels. Each of these have different subdivisions. LVA’s require a lower state of depolarization (close to resting membrane potential) in order to open, while HLA’s open and close hastily. PAGE 7
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Three channels worth mentioning are Cav1, Cav2, and Cav3 due to their important role in human physiology. Cav1 channels (otherwise known as Ltype calcium channels) have a prominent role in membrane depolarization and inducing intracellular processes in skeletal muscle cells (myocytes). Cav 1.2 and Cav1.3 serve important roles for cardiac contraction. Cav2 is mostly present in presynaptic terminals in the CNS, mediating neurotransmitter release. Lastly, Cav3 plays an important role in the heart; having a resting membrane potential close to 60mV. Mutations that affect the function of calcium channels have been related to Schizophrenia, Timothy syndrome, and intellectual disabilities.
The Voltage Gated Potassium Channels
There are different chemical classes of blockers for voltage-activated calcium channels. Phenylalkylamines, enter through the open activated channel to the intracellular pore, where it binds to a protonated specific amino acid residues sequence receptor site. The mechanism of action is both frequency and voltage dependent. Another class of blockers are the dihydropyridines, which can act as both activators and inhibitors. They work as gating modifiers and not as pore blockers. Therefore, their binding site is in a region that does not interferes with the conduction part of the channel.
Acid-Sensitive Ion Channels (ASIC) proteins are proton-gated cation channels, opening rapidly in the presence of extracellular acid. They belong to H+-gated subgroup of the degenerin/epithelial Na+ channel (DEG/ENaC) family of cation channels. ASICs are preferentially permeable to Na+, but to a lesser extent can also conduct other cations (i.e. Ca2+, K+, H+, Li+). These proton channels are found in the peripheral nervous system (PNS), where they function usually as sensory receptors, for example in specialized nerve endings (skin, muscles, intestines, heart, etc.). Their fundamental role appears to be acid transduction, converting an acidic extracellular environment into a cellular signaling event (by allowing
(VGKCs, KV) belong to a broad family of potassium channels, which play a role in the excitability of neurons by repolarization, and regulation of firing frequency so signals may transmit in the nervous system. Mutations of these channels lead to pathologies such as epilepsy, psychosis, episodic ataxia type 1 and multiple sclerosis. Peptide toxins affect these channels in two ways by interacting with the outer vestibule of the channel or the sensor domain increasing stability of the closed state. Voltage activated potassium channels are blocked by Tetraethylamonium (TEA).
Acid-Sensitive Ion Channels
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for the conduction of cations into the cell).
Multiple Sclerosis Multiple Sclerosis is a neuroinflammatory disease associated with axonal degeneration. Protongated acid-sensing ion channel-1 (ASIC1) is permeable to sodium and calcium. Excessive accumulation of these ions is associated with axonal degeneration. It is hypothesized that ASIC1 contributes to axonal degeneration in inflammatory lesions of the central nervous system. ASIC1 blockers could provide neuroprotection in multiple sclerosis. Our body’s voltage gated ion channels are extremely important in maintaining homeostasis, since an imbalance in any of these will cause different pathologies. On the bright side, years of research have unveiled channel blockers, which aid in restoring these chemical and electrical imbalances. With the increasing advance in technology and science, there may be a possibility of customizable ion gated voltage channels referenced by our genetic information.
Voltage-Gated Sodium and Calcium Channels. Molecular Pharmacology 88, 141–150. Davies, John A. “Mechanisms of action of antiepileptic drugs”. Seizure - European Journal of Epilepsy , Volume 4 , Issue 4 , 267 - 271 Heyes S, Pratt WS, Rees E, et al. Genetic disruption of voltage-gated calcium channels in psychiatric and neurological disorders. Progress in Neurobiology. 2015;134:36-54. doi:10.1016/j.pneurobio.2015.09.002. Waszkielewiez, A.M., Gunia, A., Szkaradek, N., Sloczynska, K., Krupinska, S., & Marona, H. (2013, January 18). Ion Channels as Drug Targets in Central Nervous System Disorders. Current Medicinal Chemistry, 20(10), 1241-1285. Wulff H.,Castle, N.A., Pardo, L..Voltagegated Potassium Channels as Therapeutic Drug Targets.Nat Rev Drug Discov. 2009 December ; 8(12): 982–1001. doi:10.1038/nrd2983. Zamponi, G. W., Striessnig, J., Koschak, A., & Dolphin, A. C. (2015). The Physiology, Pathology, and Pharmacology of VoltageGated Calcium Channels and Their Future Therapeutic Potential. Pharmacological Reviews,67(4), 821-870. doi:10.1124/pr.114.009654
References: Bane, Vaishali et al. “Tetrodotoxin: Chemistry, Toxicity, Source, Distribution and Detection.” Toxins 6.2 (2014): 693– 755. PMC. Catterall, W. A., Swanson, T. M. (2015). Structural Basis for Pharmacology of
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Synaptic Transmission Sebastian Brito; Giancarlo Colรณn Rosa; Hazel Cruz; Arturo Fossas; Valeria Gonzรกlez; Pedro Rivera and Robert Robinson
Introduction Nerve cells have unique features; one of those is their capability to communicate with each other with great precision through the mechanism of synaptic transmission. This mechanism is defined as the transfer of signals from one cell to another by synapses that connect one neuron with another.
Synapses Types At the present time two types of synaptic transmission are recognized in the nervous system, the electrical and chemical transmission.
Electrical Synapses In electrical transmissions, a current generated by an impulse in one neuron spreads to another neuron through a pathway of low electrical resistance. The impulse spreads via electrical synapses that occur at gap junctions. In the synapses, ion channels connect the cytoplasm of the presynaptic and postsynaptic cells with this said we could infer that electrical synapses are present where the activity of neighboring neurons need to be highly synchronized.
At an electrical synapse, the current generated by voltage-gated channels at the presynaptic neuron flows directly into the postsynaptic neuron. Therefore, transmission at such a synapse is very rapid.
Chemical Synapses In synaptic transmission at chemical synapses, there is no continuity between the cytoplasm of presynaptic terminals and postsynaptic neuron. Instead, the cells are separated by synaptic clefts, which are fluid-filled gaps. The presynaptic and postsynaptic membranes adhere to each other due to the presence of a matrix of extracellular fibrous protein in the synaptic cleft. The presynaptic terminal contains synaptic vesicles that are filled with several thousand molecules of a specific chemical substance, the neurotransmitter. Structures consisting of proteins arise from the intracellular side of the presynaptic terminal membrane and project into the cytoplasm of the presynaptic terminal. These structures and the membranes associated with them are specialized release sites in the presynaptic terminal. The vesicles containing the neurotransmitter are aggregated near these structures.
Synaptic Receptors In terms of receptors, they consist of spanning membrane proteins that have recognition sites for the binding of a chemical transmitter on its
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extracellular component. When the neurotransmitter binds to its receptor, it result in the opening or closing of ion channels on the postsynaptic membrane, which in turn activate second messengers within the postsynaptic cell.
The End-Plate In motor neurons, the cell bodies are generally concentrated in the ventral horn of the spinal cord. The neuromuscular junctions of motor axons act upon skeletal muscle fibers. There is a specialized region on the muscle membrane, the motor end plate, which is devoid of myelin and gives off branches with synaptic boutons at the end of them. Here, the muscle fiber membrane forms postsynaptic junctional folds. These boutons enclose Acetylcholine containing synaptic vessels. As the electrical stimulus reaches the terminal and depolarizes the bouton membrane, voltage-gated calcium channels open. The influx of this ion into the terminal helps the fusion of the vesicle with the terminal membrane. Consequently, the neurotransmitter for example, ACh is released by exocytosis. The action of ACh on the nicotinic cholinergic receptors and the subsequent postsynaptic potential in the muscle fiber is referred to as an End Plate Potential (EPP).
The End-Plate Potential In the End Plate Potential, there is an inflow of sodium and an outflow of potassium out of the postsynaptic cell due to the bipermeability of the ionic channel to both Na and K. Afterwards, acetylcholinesterase inactivates ACh by cleaves it, and it is later reabsorbed by the nerve terminal as its constituents to be later reused. In directly gated transmission at a central synapse, graded potentials are produced, which are defined as local changes in neuronal dendrite and cell body membrane potentials (and not in axons). Their amplitude is proportional to the intensity of the stimulus. These potentials travel through the neuron until they reach the trigger zone, which is located at the axon hillock in efferent neurons. If they reach the threshold potential, and all-or-nothing action potential is triggered.
Summation As multiple signals at the trigger zone superimpose, we achieve the phenomenal of summation. There are 2 types of summation: spatial and temporal summation. In spatial summation, many different presynaptic neurons send potentials and cause the postsynaptic neuron to fire as it reaches threshold. In temporal summation, there is only one presynaptic neuron firing many times in rapid-succession, which causes the postsynaptic neuron to fire as it PAGE 11
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reaches its threshold. Several substances can inhibit a neuron from reaching threshold potential by producing a hyperpolarizing graded potential that drives the potential away from firing. Such a potential is called an inhibitory postsynaptic potential, and examples of such inhibitory neurotransmitters are GABA and glycine.
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Miniature End-Plate Potentials Jamilisse Segarra; Ashley Soler; Nicole Rivera; Frank Zorrilla; Javier Sevilla; Alexandra Vega and Manuel Rovira
Introduction Miniature end plate potentials (MEPPs) are small spontaneous depolarizations that occur in the postsynaptic terminal1. MEPPs are caused by a discrete, irregular release of acetylcholine. Abnormal Miniature End-Plate Potentials are correlated with neuromuscular dysfunction2. Compound spatial summation of Miniature End-Plate Potentials (MEPPs) creates End-Plate Potentials (EPPs) and activates muscle contraction.
Methods The method utilized for the delivery of the material regarding “Miniature End-Plate Potentials” was an in-class oral power point presentation followed by an open dialogue for any necessary clarifications or further explanation.
Discussion Presynaptic terminals spontaneously release acetylcholine at a minimal degree causing a Miniature End-Plate Potential (MEPP). MEPPs are random, small depolarizations (less than 1 mV)
due to acetylcholine, but not enough to reach threshold and depolarize the postsynaptic membrane. In most circumstances, more than one vesicle of acetylcholine will be released upon stimulation. The MEPPs resulting from each vesicle will summate, form an end-plate potential (EPP) and bring the postsynaptic membrane to threshold, leading to an action potential. An MEPP will not trigger an action potential in the muscle, however an EPP will. Presynaptic acetylcholine release binding to its postsynaptic receptor depolarizes the muscular membrane near the neuromuscular junction and causes both, motor end-plate potentials and associated “noise” 3. MEPPs have been studied in neuromuscular diseases, most notably in the search for clinical treatments for Myasthenia Gravis (MG). MG results from an autoimmunemediated attack on the postsynaptic acetylcholine receptor of muscle cell membrane. Experimentally, the amplitudes of MEPPs and EPPs are lower when compared to normal subjects3 .The pathological antibodies that block the neuromuscular junction (NMJ) are the main target of treatment. These include the following: (1) reduction complement system attack, as well as antibody production, (2) curbing of dendritic cell activity in antibody production (3) improved of AChR strength with stabilizing proteins, and (4) blatant removal of the excess autoantibodies from patient serum. Many of these PAGE 13
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tactics provide temporary relief and have yet to show definite signs of long term results; although researchers remain optimistic 4. Botulism is a clear case of the importance of acetylcholine as the neurotransmitter involved in MEPP. If the toxin produced by Clostridium botulinum reaches presynaptic neuron’s cytoplasm, it will destroy docking mechanisms that cholinergic vesicles utilize, thus disrupting their exocytosis. Therefore, amplitude and frequency of MEPP will decrease since there will be no acetylcholine inside the vesicles being exocytose into the synaptic cleft. In case that there has already been neuronal damage, the available treatment is an antitoxin that should be administered as soon as possible. However, treatment will not reverse the neuroparalysis that has already occurred, thus requiring mechanical ventilation in order to prevent respiratory arrest 5, 6.
Conclusion MEPPs provide a multitude of improvement possibilities of current incurable neuromuscular diseases, like myasthenia gravis and botulism. With profound understanding of these almost minute electrical stimuli, further research could open the gate for new ways to manipulate neuromuscular firing. The MEPPs provide a possible “back up” system in scenarios where action potentials are otherwise lost or hindered; and the research looks to capitalize on them
aiming to better the quality of life for patients with these difficult, neuromuscular disorders.
References Johnson, L. R., & Byrne, J. H. (2003). Essential Medical Physiology. Academic Press. pp 108-110 Klooster, R. et al. “Muscle-specific kinase myasthenia gravis IgG4 autoantibodies cause severe neuromuscular junction dysfunction in mice”. Brain 2012. 135 (4): 1081-1101. Daube, J. R., & Rubin, D. I. (2009). Clinical neurophysiology. Oxford: Oxford University Press p.98,370-380 Mantegazza, Renato et al. “Current and Emerging Therapies for the Treatment of Myasthenia Gravis.” Neuropsychiatric Disease and Treatment 7 (2011): 151– 160. PMC. Web. 23 Mar. 2017 Mantilla, C. B., Stowe, J. M., Sieck, D. C., Ermilov, L. G., Greising, S. M., Zhang, C., . . . Sieck, G. C. (2014, October 15). TrkB kinase activity maintains synaptic function and structural integrity at adult neuromuscular junctions. Retrieved March 21, 2017, from http://jap.physiology.org/content/117/8 /91 Office of Public Health Preparedness and Response (OPHPR). (2006, June14). CDC Botulism. Retrieved March 20, 2017, from https://emergency.cdc.gov/agent/botulis m/clinicians/treatment.asp
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Patch Clamp Methods and Applications Gabriela Fenollal Maldonado, Luis Cruz Saavedra, María del Mar Meléndez González, Andrea Otero Ríos, Santos Santos Fontánez, Amin Hussein Hassan and Orlando Rivera Estrada
applied in order to create a seal between the 2 surfaces. In this way, ion exchange measured comes only from the channel being examined. This ionic current is measured using an amplifier attached to the pipette that can detect if that channel is open or closed and it can be used to compare different channel activities.
Applications The Patch Clamp Technique The technique of membrane voltage fixation is a laboratory procedure that allows studying the electrophysiology of one or more ion channels in the cell. It was invented and developed by Bert Sakmann and Erwin Neher, winners of the Nobel Prize in Physiology and Medicine in 1991, in the mid-1970s. This discovery was the beginning of understanding how the ion channels in the membrane are involved in action potentials and nerve activity. By electrically isolating a patch of membrane from its surrounding solution, it allowed the study of the ion channels on the cell membranes, their changes in currents, malfunctions and the effects of drugs on these ion channels. Physiologically speaking, this can be observed in a number of cells found in the human body such as neurons, cardiomyocytes, and muscle fibers. This technique makes use of a glass pipette that comes into contact with the neuronal membrane. Once contact is made, a small amount of suction is
The industrial and academic sectors are enhancing the patch-clamp processes due to the demand for accurate data production. During the last couple of decades, research has found that many diseases are caused by aberrations in the ion channels and new medications are being developed. At least 10% of the drugs that are distributed in the market target ion channels and these have a value of $10 billion. Many well-renowned medications that target ion receptors are being marketed for many conditions such as: antihypertensives and antihyperaritmics. Also drugs are being used to treat stroke, epilepsy, localized pain and muscular tension. Each one of these medications have differences on their mechanism of action that make them selective to certain ion receptors. The development of the patch-clamp method made possible to record the currents of single ion channel molecules. This advancement improved the understanding of the roles of different ion channels in
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fundamental cell processes (ex. Action potentials). It has special importance in the investigation of excitable cells (such as neurons and cardiomyocytes) and live-cell imaging (such as Ca2+). In medical research, it has been used to detect malfunctions in ion channels that are the cause of several diseases. Finally, in pharmacological research, automated patch clamping is used to screen potent substances for ion channel modifications. Some of the disadvantages of the technique is that in some of the procedures the resting membrane potential and composition of ICF and ECF cannot be controlled; important intracellular components may be replaced by the contents of the electrode and that measurement of ion currents from individual channel openings may not be possible, like in the Whole Cell procedure.
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Hypokalemic Periodic Paralysis Cristina Cardona; Teresa Del RĂo; Reynat Jimenez; Jean Paul Godreau; Eduardo Nogales and Zoe Underill
Introduction: Hypokalemic periodic paralysis (HOKPP) is a condition in which affected individuals may experience paralytic episodes characterized by low levels of potassium in their blood. The person experiences muscle weakness and inability to move its extremities for several hours or sometimes days. The frequency of the attacks can vary from one in a lifetime to daily, monthly or less often. Some factors that may trigger the onset are carbohydrate-rich meals and rest after exercise, a viral illness, and certain medications and rarely, cold-induced hypokalemic paralysis can occur.
Methods GENETICS
HOKPP can be caused by a mutation in several genes. The two principal mutations that can cause this condition are loss-of-function mutations in the CANA1S (a voltagegated calcium channel found in the transverse tubules of skeletal muscle cells) and in SCN4A (a voltage-gated sodium channel found at the neuromuscular junction). Both mutations would result in a decreased responsiveness of the muscle to
neural stimulation, resulting in an inability of the muscle to contract and, paralysis. The resulting hypokalemia further aggravates the muscle responsiveness, leading to an even more severe paralysis. DIAGNOSIS
In the diagnosis of primary hypokalemic periodic paralysis, for clinical findings to be used as diagnostic factors there must be a familial history of the disease. In addition, to family history there are several criteria of clinical symptoms that must be met for an indicative diagnosis. First and foremost, any causes of hypokalemia not related to the disorder must be excluded. Additionally, the patient must have a history of more than two episodes of muscle paralysis attacks with a plasma potassium concentration less than 3.5 mEq/L. If the patient has only had one previous attack of muscle paralysis, then they must have a family member that has also had a previous attack with a documented plasma potassium concentration less than 3.5 mEq/L. If there is no history of previous attack then at least three of the following six clinical findings must be met. Attack occurring in first or second decade; attack lasting more than two hours; the identification of triggers (i.e. resting after exercising, eating a meal high in carbohydrates, stress, prolonged paralysis) causing the attack; symptom improvement after exogenous potassium ingestion; family history of condition; and/or a
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positive long exercise test. Nonclinical diagnosis may be achieved through molecular genetic testing of genes, CANA1S, SCN4A, and, KCNJ18. These genes encode subunits of ion channels in skeletal muscle cells and hypokalemic periodic paralysis may be attributed to their mutation. (Vicart et al., 1993)
Discussion DIFFERENTIAL DIAGNOSIS
HOKPP can be distinguished from other types of hypokalemia by a number of factors. For one, ensure the patient is not taking any prescription or over-the-counter medications such as laxatives or alcohol, which may lead to potassium wasting. Additionally, ensure that the patient does not have any other condition such as hyperaldosteronism, Conn syndrome, or diarrhea, which may also lead to potassium wasting. Urinary potassium wasting can be measured using a urine sample and will help in distinguishing urinary potassium wasting (if urinary potassium concentration >20 mmol/L) and intracellular shift of potassium, as is seen in HOKPP. Levels of TSH, triiodothyronine and thyroxine should be measured, as thyrotoxicosis may be a causation of periodic paralysis. Viruses and tick bites may also cause periodic paralysis, and history of either of these things would indicate against HOKPP as the primary diagnosis. Additionally, symptoms such as ptosis, diplopia, and dysphagia
in addition to the episodic paralysis would suggest an involvement of the neuromuscular junction rather than HOKPP. TREATMENT
During attacks, oral potassium supplementation is preferable to IV supplementation. Potassium chloride is the preferred agent for an acute attack. Typically, one should not exceed a total dose of 200 mEq in a day. Intravenous potassium is reserved for cardiac arrhythmia or airway compromise due to ictal dysphagia or accessory respiratory muscle paralysis. Mannitol should be used as solvent, as both sodium and dextrose worsen the attack. Approximately 50% of genotyped patients with HOKPP respond to acetazolamide. Poor response is predicted with substitution of arginine with smaller glycine in the residues of voltage sensors near the extracellular side of the sarcolemma.
Conclusion Hypokalemic periodic paralysis (HOKPP) is a condition that is autosomal dominant; a parent usually is a carrier. The amounts of de novo mutations are not known since little research has been done on the matter. The condition is known to cause a series of attacks that leave the person on a flaccid state, the paralysis mostly affects the muscles of the arms and
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legs but during severe attacks, it might also affect trunk muscles, which could lead to problems with swallowing or breathing. On the moment of the attack, potassium is low but in between attacks, it is maintain on normal levels. The ideal treatment or prevention is maintaining the patient on a low carb diet; avoid fatty foods, and alcohol. It is important to mention that the onset of this condition is after puberty, first with small attacks and then it progresses on to more severe attacks. When a person goes into this paralysis the treatment is potassium shot, and beware that the person is fully conscious at all times.
Vicart S, Sternberg D, Arzel-Hézode M, et al. (1993). Hypokalemic Periodic Paralysis. In R. A. Pagon, M. P. Adam, H. H. Ardinger, S. E. Wallace, A. Amemiya, L. J. H. Bean, T. D. Bird, N. Ledbetter, H. C. Mefford, R. J. H. Smith, & K. Stephens (Eds.), GeneReviews(R). Seattle (WA): University of Washington, Seattle
Works Cited HypokalemicPeriodic Paralysis - Genetics Home Reference. (2007, April). Retrieved March 21, 2017, from https://ghr.nlm.nih.gov/condition/hypok alemic-periodic-paralysis#genes Greant, D. C. (2011, September). Do I Have Periodic Paralysis. Retrieved March 21, 2017, from http://hkpp.org Rüdel R, Lehmann-Horn F, Ricker K, Küther G (February 1984). "Hypokalemic periodic paralysis: in vitro investigation of muscle fiber membrane parameters". Muscle Nerve. 7 (2): 110–20. doi:10.1002/mus.880070205. PMID 6325904. Tachamo, N., & Lohani, S. (2017, January 30). CASE REPORT Paralysis that easily reverses: a case of muscle paralysis. Retrieved March 22, 2017, from
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Cystic Fibrosis José L. Ortiz Fullana; Cristina Cruz; Edgardo Rivera; Gabriela Rosario; Omar Rodríguez; Natalia Del Mazo and José Nuñez
Introduction Transmembrane Conductance Regulator (CFTR) is found in epithelial cells of the lungs. Normally, this protein moves chloride ions out of the cell to the covering mucus causing sodium ions to follow passively, resulting in the movement of water out of the cell via osmosis making the mucus thinner. Cystic fibrosis (CF) is a disease caused by a deletion of 3 nucleotides from the gene that encodes the CFTR. As a result of this mutation, there is an inability to transport chloride across epithelial cells leading to decreased secretion of chloride ions and increased reabsorption of sodium and water, resulting in the production of thick mucus in the lungs and increased susceptibility to infections.
Methods Cystic Fibrosis symptoms vary depending in the severity of the disease and some people may not present symptoms until youth or adulthood. Respiratory symptoms, which are caused by the thick mucus that clogs the airways, includes persistent cough, wheezing, breathlessness and lung infections. The gastrointestinal symptoms are
also related block tubes that carry digestive enzymes from pancreas to small intestine causing inability completely absorb nutrients from food. Digestive symptoms include, greasy stools, poor weight gain and growth and severe constipation. The CFTR can be found on the pancreatic and bile ducts’ epithelium and small intestine while; it is almost nonexistent on the colon. The presence of these defective CFTRs on the pancreatic and biliary ducts prevent the secretion of bile and pancreatic digestive enzymes. The absence of bile and pancreatic enzymes results in poor fat and nutrient absorption and therefore weight loss and greasy stools. On the other hand, the excessive thick mucus on the gastrointestinal tract may form mucus plugs resulting in constipation.
Discussion There is no cure for CF, but certain treatments and medications can reduce the symptoms and complications of the disease. Some medications include antibiotics, antiinflammatory drugs, mucus-thinning drugs, inhaled medications and oral pancreatic enzymes. Moreover, the patient may receive chest physical therapy and pulmonary rehabilitation. On the other hand, in some cases surgery may be needed to relieve some symptoms like airway obstruction and nasal polyps. More importantly, CF has been at the forefront of clinical trials involving
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gene therapy. However, the lungs are a difficult target to treat due to their ability to fight off invasions of foreign particles, which is a barrier, that Gene Therapy Agents (GTAs). Recently, in 2012, Kalydeco (ivacaftor) was released to the market and has given proof of concept that correction of molecular defects leads to clinical benefit. Future studies are focusing in the possibility of using non-viral vectors (GTAs) in order to correct chloride transport. Non-viral vectors (GTA) will not produce immune responses and will be consistently effective after repeated administration. A multi-dose clinical trial with cationic lipid GL67A has been investigated and a safe dose suitable for progression was identified. Next, is a double-blinded placebo controlled study is needed to determine if there is a change in baseline %FEV1, lung clearance improvement and quality of life.
Conclusion CF is an autoimmune disease that remains as a primary target for gene therapy, which has been proven to be successful. However, it requires much more research to determine the adequate gene therapy and dosage. On the other hand, the scientific community has a great understanding of its pathophysiology, which is the stepping stone of any scientific breakthrough.
References Chandar, Nalini. Lippincott's Illustrated Reviews: Cell and Molecular Biology. N.p.: Lippincott, Williams & Wilkins, 2010. Web. Gastrointestinal Manifestations of Cystic Fibrosishttps://www.ncbi.nlm.nih.gov/p mc/articles/PMC4865785/ Griesenbach, U., & Alton, E. (2013). Moving Forward: cystic fibrosis gene therapy. Human Molecular Genetics,22(1), 52-58. doi:10.1093/hmg/ddt372 Intestinal Obstruction Syndromes in Cystic Fibrosis: Meconium Ileus, Distal Intestinal Obstruction Syndrome, and Constipation https://www.ncbi.nlm.nih.gov/pmc/artic les/PMC3085752/ Lyczak, J. B., Cannon, C. L., & Pier, G. B. (2002). Lung Infections Associated with Cystic Fibrosis. Clinical Microbiology Reviews, 15(2), 194–222. http://doi.org/10.1128/CMR.15.2.194222.2002 What is cystic fibrosis? National Heart, Lung, and Blood Institute. http://www.nhlbi.nih.gov/health/healthtopics/topics/cf. Accessed July 1, 2016.
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Hyperkalemic Periodic Paralysis Agnes Diane León; Laura Bimbela; Laura Colón; Luis J. Martínez; Fabiola A. Morales Vias; Mariluz Ramos; Ricardo Vallejo
Introduction Hyperkalemic Periodic Paralysis (HyperPP) is an autosomal dominant muscle disorder, which causes intermittent episodes of sudden muscle contractions or twitches and subsequent paralysis. It is caused by a point mutation in the SCN4A gene and has been estimated that 1 in 200,000 people are affected. This disorder has been linked to diets high in potassium and other environmental factors such as stress, cold and fatigue. These factors can trigger sudden attacks of muscle weakness and myotonia (muscle stiffness) which can be infrequent and mild in early onset of the disorder but later it may progress in frequency and severity. Latest research has shown that a functional polymorphism may be involved in the severity of the disorder, causing enhanced defects in a novel mutation in the Nav1.4 channel, which can lead to greater clinical manifestations in the patient.
Methods To gather information about Hyperkalemic Periodic Paralysis we researched literature in websites such
as NCBI, UpToDate, NIH, and prepared a PowerPoint presentation.
Discussion Hyperkalemic Periodic Paralysis is a condition characterized by weakness in skeletal muscles. The principal cause of this condition is a mutation in the SCN4A gene, which codes for a sodium transporter in skeletal muscle membranes. Many factors such as, ingestion of food high in potassium (like bananas and potatoes), pregnancy, stress, rest after exercise, and others can trigger the episodes of this condition. To diagnose this disorder the patient’s symptoms and his/her family history is evaluated. A genetic test is employed to look for and analyze the SCN4A gene. In addition, laboratory findings show elevated serum potassium levels (> 5 mmol/L or an increase of 1.5 mmol/L). An electrocardiogram can be abnormal and present a T wave with increased amplitude. Another important test is an electromyocardiogram, which would show less functional motor units or some that may be silent. If after performing the routine tests there is still uncertainty, alternative provocative tests are indicated. The doctor performing these exams must take into consideration the patient’s clinical picture because inducing an attack may be particularly dangerous. The onset of an attack can be prevented with sugar and mild exercise to increase K uptake. Often, no treatment is needed due to its brief
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nature. Severe attacks can be treated with thiazide diuretics that inhibit NaCl- cotransporter located at the distal tubule. This increases the Na reabsorption of principal cells located at the distal tubule and collecting duct, stimulating K secretion. Also, Albuterol (1 or 2 pumps 0.1mg), a beta-adrenergic agonists is used because it promotes cellular reuptake of potassium by stimulating the Na/K ATPase pump. In more severe attacks, intravenous calcium may be administered as a preventive measure against cardiac rhythm disturbances. Recent studies have found a mutation in the Nav1.4 channel, I692M, in genes of identified HyperPP families. Along with this mutation, in some of these families a known polymorphism S906T was also present. The authors observed that S906T enhanced the effects of the mutation I692M, patients with I692M-S906T appeared to be more affected than those who only had the I692M mutation. They found that patients with I692M-S906T had longer weakness episodes, their muscles were more affected, CK was elevated and there was presence of permanent weakness (Fan C. et al, 2016).
higher than normal level of potassium in the blood. These periodic episodes can be provoked by many different stresses; keeping track of its past triggers serves as a good preventive measure.
References http://hkpp.org/patients/hyperkpp-FAQ https://medlineplus.gov/ency/article/00 0316.htm Fan, C., Mao, N., Lehmann-Horn, F., BĂźrmann, J. and Jurkat-Rott, K. (2016), Effects of S906T polymorphism on the severity of a novel borderline mutation I692M in Nav1.4 cause periodic paralysis. Clin Genet. doi:10.1111/cge.12880 Weber F, Jurkat-Rott K, Lehmann-Horn F. Hyperkalemic Periodic Paralysis. 2003 Jul 18 [Updated 2016 Jan 28]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviewsÂŽ [Internet]. Seattle (WA): University of Washington, Seattle; 19932017. Available from: https://www.ncbi.nlm.nih.gov/books/NB K1496/
Conclusions Hyperkalemic Periodic Paralysis is a genetic disorder of impaired muscle contractions characterized by an autosomal dominant mutation in the SCN4A gene. It causes occasional episodes of muscle weakness and sometimes a
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Malignant Hyperthermia
receptors. This accumulation of calcium causes sustained muscle contraction.
Luisa Bernacet Rivera; Luis M. Cabrera; Monica Montes; Carolina RodrĂguez Rivera; Miguel Santiago Cruz; Coralys Soto and Katiana M. Vazquez
Triggers
Molecular Background Malignant hyperthermia (MH) is a lifethreatening hypermetabolic syndrome caused by the exposure to volatile anesthetics. It is an autosomal dominant gain-of-function mutation in skeletal muscle ryanodine receptors (RyR1), a calcium-gated calcium channel in the sarcoplasmic reticulum membrane. Its main function is to increase the calcium concentration intracellularly when stimulated by a small calcium influx through the dihydropyridine receptor situated in the T-tubule membrane. It is believed that the mutation is due to a substitution of the amino acid glycine for arginine at position 2434. This mutation interrupts the binding of FKBP cytoplasmic protein with the cytoplasmic domain of RyR1, making the receptor hypersensitive to calcium. The intracellular calcium increase causes the excitationcontraction coupling which initiates muscle cell contraction. Specific agents leading to an unregulated release of calcium from the sarcoplasmic reticulum to the intracellular space trigger mutations encoding for abnormal ryanodine
The majority of cases of malignant hyperthermia have occurred while the patient was receiving a volatile anesthetic agent (halothane, enflurane, isoflurane, sevoflurane). Other cases of malignant hyperthermia have been reported by the use of the muscle relaxant succinylcholine. Patients susceptible to MH may not develop the acute syndrome every time they are exposed to these agents, history of exposure prior attack may be present.
Clinical Presentation Clinical manifestations of malignant hyperthermia may vary between patients and may present perioperatively in several possible patterns and degrees. However, despite variability, clinical signs tend to present in a particular order. One of the earliest sign of the condition is a Masseter spasm shortly after administration of the triggering agent. Other early signs include hypercarbia (resistant to minute ventilation), sinus tachycardia, and a generalized muscle rigidity despite the administration of neuromuscular blockers. However, hypercarbia is the most common initial sign among patients. On the other hand, hyperthermia is often a later sign along with
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ventricular fibrillation, ventricular bigeminy, and excessive bleeding. Typical laboratory findings confirmatory of acute MH elevated serum levels of creatinine kinase, potassium, and myoglobin; all signs of rhabdomyolysis. Another common finding is a mixed metabolic and respiratory acidosis caused by elevated blood gas PaCO2 and lactic acid from excess glycolysis in muscle
Treatment Malignant hyperthermia protocol established by the Malignant Hyperthermia Association of the United States are as follow:
MONITOR AND TREAT HYPERKALEMIA
References Litman R. Malignant hyperthermia: Clinical diagnosis and management of acute crisis [Internet]. 2016 [cited 2017 Mar 22]. Available from: http://www.uptodate.com/contents/mali gnant-hyperthermia-clinical-diagnosisand-management-of-acute-crisis U.S. National Library of Medicine, Genetics Home Reference. RYR1 Gene: ryanodine receptor [Internet] 2016 [cited 2017 Mar 22]. Available from: https://ghr.nlm.nih.gov/gene/RYR1#
OPTIMIZE OXYGENATION AND VENTILATION- Increase inspired oxygen
to 100%, ventilation rate and/or tidal volume to maximize ventilation and reduce the ETCO2. Patient should be intubated. DISCONTINUE TRIGGERING AGENTS-
Discontinue anesthetic agents immediately and inform the surgeon. Surgical procedures should be stopped as quickly as possible. If surgery cannot be stopped, it should be completed under intravenous anesthesia with non-triggering agents. ADMINISTER DANTROLENE- Dantrolene,
the only known antidote for MH, should be administered as loading amounts of 2.5mg/kg intravenously (IV), with subsequent doses of 1 mg/kg IV until signs of MH have subsided.
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