Myotonia Congenita is a genetic disorder that is characterised by the inability of skeletal muscle to relax after voluntary movement[1]. Individuals who are diagnosed with different forms of myotonia congentia typically describe a painless, muscle stiffness that remits with several repetitions of the same movement [2]. Myotonia is caused by mutations within the CLCN1 gene that decreases the chloride channel activity in the muscle in turn prolonging potential equalisation during voluntary muscle movement. The activation of chloride ion channels are essential for ensuring the electrical stability of skeletal muscle by resetting its membrane excitability to the resting state after firing an action potential [4]. There have been suggestions that Myotonia may cause infertility in males [3]. However, it is still relatively unclear how myotonia and infertility are related.
My long-term goal is to understand how myotonia congenita affects chloride channel activities that lead to infertility in males. My primary goal is to determine how the CLCN1 gene regulate chloride ions in testis. My hypothesis is that mutations in the CLCN1 gene decreases the transmembrane transport of chloride ions in turn affecting the electrochemical gradient causing sperms to be immobilised. I will use mice as my model organism because they share similar muscle structure, reproductive system, and have skeletal muscle movements that are easy to observe.
Aim 1: Determine conserved amino acids in the CLCN1 protein that mediate proper sperm function. Approach: ClustalOmega will be used to determine specific amino acids in CLCN1 that are responsible for chloride transport in testis. CRISPR/Cas9 will then be used to induce mutation within the CLCN1 gene of male mice. Knockouts that result in infertility in the male mice will then be tested for proper sperm function Rationale: Since the CLCN1 gene is expressed mainly in the skeletal muscle and only a some in testis. Identifying sequences that are responsible for the chloride channels in testis will allow us to differential different chloride channels. Hypothesis: I expect alleles with mutations will cause infertility in males.
Aim 2: Determine gene expression changes necessary for fertility in CLCN1 mutant mice Approach: RNA-seq will be conducted on testis tissue samples from both WT and WT knockout mice generated in Aim1. I’ll then analyse gene expression levels between wild type and CLCN1 mutants. Genes with different expression specific to CLCN1 mutant testis tissue will be identified. I will then use PANTHER to determine what Gene Ontology are differentially expressed in infertile mice Rationale: Understanding the gene expression patterns of Chloride channel genes specifically in the testis can provide insight into the role of CLCN1 in testis. Hypothesis: Mutations in CLCN1 will result in decreased activity of chloride channel within the testis thus disputing the mobility of the sperms.
Aim 3: Identify novel CLCN1 protein interactions important for sperm development Approach: I will perform a Tandem affinity purification- mass spectrometry on testis of adult wild type and CLCN1 knocked out mice. I will compare the protein interactors among all samples and isolate the proteins that can interact with wild type mice but not the domain mutants. I will then identify the interactors by mass spectrometry. Rationale: CLCN1 protein interactions can arise from co-existence in organised structure arrangement such as membrane proteins. The introduction of specific stable chemical linkages can establish other unknown factors that affects the mobility of sperm cells. Hypothesis: I expect to see abnormal (newly) protein interactions in the CLCN1 knockout mice.
[1] Colding-Jørgensen E. Phenotypic variability in myotonia congenita. Muscle Nerve. 2005 Jul;32(1):19-34. Review. [2]Chrestian N, Puymirat J, Bouchard JP, Dupré N. Myotonia congenita--a cause of muscle weakness and stiffness. Nat Clin Pract Neurol. 2006 Jul;2(7):393-9; quiz following 399. [3] Trip J, Faber CG, Ginjaar HB, van Engelen BGM, Drost G. Warm-up phenomenon in myotonia associated with the V445M sodium channel mutation. Journal of Neurology. 2007;254(2):257-258. doi:10.1007/s00415-006-0353-2. [4] A. F. Dulhunty, “Distribution of potassium and chloride permeability over the surface and T-tubule membranes of mammalian skeletal muscle,” Journal of Membrane Biology, vol. 45, no. 3-4, pp. 293–310, 1979. [5] Extraction, Enrichment, Solubilization, and Digestion Techniques for Membrane Proteomics Stephanie M. Moore, Stephanie M. Hess, and James W. Jorgenson Journal of Proteome Research201615 (4), 1243-1252 DOI: 10.1021/acs.jproteome.5b01122