Hello my name is maryvi gonzalez im from the university of PR @ cayey, from the general natural sciences department. This summer I have the opportunity to work under the mentorship of Dr. William Atchison and Elizabeth Molina-Campos, both from MST, dptmt of Pharm. & Tox. This project is sponsored by the Ronald E. McNair Scholars Program. The tittle of my research is The Effects of P/Q type ca channels subunits alpha 1 a and their replacements by N and R type ca channels.
The NMJ are the relation between a somatic motor neuron, which is the presynaptic terminal of the neuron and the muscle fiber also known as the postsynaptic membrane. Neurons communicate with muscle releasing neurotransmitters, in this case Ach. When Ach is released, this indicates the muscle to contract. Ach release is triggered by calcium entrance into the neuron, because of VGCC and most concentration of Ca aoutside the cell. Once Ach is released by the vescicles, , travels through the synaptic cleft and is received by Ach receptors on the postsynaptic membrane.
As we see, VGCC are important in the release of Ach. There are many subtypes of calcium channels, but this research is concentrated in the P/Q type ca channels. These Ca channels are compound of many subunits, but the main subunits are named alpha 1 a. Other subunits of this ca channels are beta, gamma, delta and alpha 2 delta, which have the role of modulating the alpha 1 a activities. These ca channels are voltage gated because they are opened when theres a change in membrane potential, which permits the selective entrance of calcium into the neuron.
In LEMS autoantibodies target mainly the alpha 1 a VGCC by blocking them and this does not permit the entrance of calcium into the neuron, which does not permit the release of Ach and affects muscle contraction.
The incidence of LEMS is between 17 to 79 years old, predominantly in males than females. The common symptoms are skeletal muscle weakness, decreased tendon reflexes and dysautonomias (a breakdown or failure in the autonomic nervous system). Also patients can have fatigue, body weakness, light headiness and cognitive impairment (boundary or transition stage between normal aging and dementia-wikipedia.org).
This research involve background from two specific studies: Flink’s which describes that WT mice were treated with LEMS plasma during 30 days and he unmasked the prescence of L-type Ca channels, to compensate the loss of P/Q-type ca channels. Pardo’s research, she found out that in Tg mice, N and R type ca channels appears to replace the P/Q type ca channels.
Based on these two conclutions, I have the pourpose of this research, to determine if LEMS leads to unmask other subtype of Ca channel in TG mice.
As materials and methods, the mice that are on test are the WT mice, which is normal and the Tg mice, which is a mice that genetically came with damage in the P/Q type calcium channel, alpha 1 a subunits, which means that is not functional. The antibodies Im using are against specific calcium channels subunits, like the alpha 1 a which is for P/Q type, alpha 1 b which is for the N type, alpha 1 c, which is for the L-type and the alpha 1 e which is for the R-type. The two techniques for this research are western blot, where I extract proteins from front mice legs and blot them with antibodies to observe what calcium channels subunits are unmasked, and immunohistochemistry
Effects of LEMS on P/Q type Calcium Channels
The effects of LEMS on P/Q-type Ca ² + channel α 1 A subunit and its replacements by N- and R-type Ca ² + channels. Maryví González ¹ , Elizabeth Molina-Campos ² and William D. Atchison ² Universidad de Puerto Rico at Cayey, Department of General Natural Sciences ¹ Michigan State University, Department of Pharmacology & Toxicology ² Ronald E. McNair Post-Baccalaureate Achievement Program Research Initiative for Scientific Enhancement Program
Introduction <ul><li>Neuromuscular Junctions (NMJ) </li></ul><ul><li>Acetylcholine (ACh) </li></ul><ul><li>Voltage-gated Ca ² + Channels (VGCC) </li></ul>http://www.colorado.edu ACh Ca² + Ca² +
http://www.cellscience.com http://www.biochem.ubc.ca Figure 2 Ca ² + Ca² + Voltage gated Ca 2+ channels P/Q-type
Introduction Continued <ul><li>Lambert-Eaton Myasthenic Syndrome (LEMS). </li></ul><ul><li>Autoantibodies target the VGCC that regulates ACh release at motor nerve terminals (mainly α 1 A ). </li></ul>AB
Introduction Continued <ul><li>Incidence: </li></ul><ul><ul><li>60% has SCLC (small cell lung cancer) </li></ul></ul><ul><ul><li>Range from 17 to 79 years old </li></ul></ul><ul><ul><li>Males are more predominantly affected than females </li></ul></ul><ul><li>Symptoms </li></ul><ul><ul><li>Skeletal muscle weakness </li></ul></ul><ul><ul><li>Decreased tendon reflexes </li></ul></ul><ul><ul><li>Malfunctions of the autonomic nervous system </li></ul></ul>
Background <ul><li>Genetic mutations has been described for several types of calcium channels, including the P/Q-type. </li></ul><ul><li>One example of the mutation is tottering ( tg) mouse. </li></ul><ul><li>The tg mice mutation encodes a proline-to-leucine amino acid substitution in the S5-S4 linker region of repeat domain II of the α 1A subunit. These subunits are not functional. </li></ul><ul><li>WT mice normally release ACh to ACh receptors in muscle fibers, which is triggered by calcium, that enters through P/Q-type Ca² + channels. </li></ul>
Background <ul><li>In LEMS mice there is reduced P/Q-type calcium channels, but other types of calcium channels appear to compensate . </li></ul><ul><li>WT mice treated with LEMS plasma for 30 days leads to compensation by unmasking of L-type Ca² + channels in ACh release to compensate for the loss of functional P/Q-type Ca² + channels. (Flink, 2002) </li></ul><ul><li>In adult tg compensation does not involve L-type calcium channels, but instead involves N- and R-type Ca² + channels which assume control of ACh release at motor nerve terminals. (Pardo, 2006) </li></ul>
Question: <ul><li>In tg mice treated with LEMS plasma, what other calcium channels subtypes can compensate the loss of P/Q-type calcium channels? </li></ul>
Purpose <ul><ul><li>Determine if treatment with LEMS plasma leads to an unmasking of other subtypes of Ca ² + channels in tg mice. </li></ul></ul>
Materials & Methods <ul><li>Mice: </li></ul><ul><ul><li>tg Mice = Tottering Mice </li></ul></ul><ul><ul><li>wt Mice = Wild Type Mice </li></ul></ul><ul><ul><li>LEMS plasma from two patients </li></ul></ul><ul><li>Western Blot </li></ul><ul><ul><li>Antibodies: </li></ul></ul><ul><ul><li>α 1 A (P/Q-type), α 1 B (N-type), α 1 C (L-type), α 1 E (R-type) </li></ul></ul>
Results tg wt sal. tg +/+ sal. IRC um1 tg +/+ um1 tg +/+ PJ tg wt PJ a. 230 kDa. b. 220 kDa. b. 75 kDa. c. 148 kDa. d. 180 kDa. e. β -Actin α 1 B α 1 C α 1 C α 1 E α 1 A
Conclusion <ul><li>Antibodies to α 1A , and α 1E subunits obtained from Alomone Labs displayed low specificity, since the molecular weight of the proteins stained by them was not the predicted one. In α 1B and α 1C , revealed bands at the appropriate MW. </li></ul><ul><li>Apparently there is an expression of other subtypes of Ca 2+ channels in tg mice as α 1C , which appeared to be expressed in tg +/+ LEMS 1 and tg +/+ LEMS 2, but different percentage of change between these are observed. In order to confirm this conclusion, more trials have to be done and calculate the standard mean of error for the values obtained. </li></ul>
Acknowledgments <ul><li>Dr. William D. Atchison- Mentor </li></ul><ul><li>Elizabeth (Pampa) Molina-Campos- Proxy </li></ul><ul><li>Brenda Marrero Rosado </li></ul><ul><li>McNair Staff & Dr. Roop Jayaraman </li></ul><ul><li>Fellow McNair Scholars </li></ul><ul><li>Special thanks: mother, father, grandmother </li></ul>