UTILIZING C. ELEGANS AS A NEUROLOGICAL MODEL TO CHARACTERIZE KCNL-2, AN SK CHANNEL HOMOLOGUE

In the mammalian nervous system, SK channels function to regulate neuronal excitability through the generation of a component of the afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson’s disease. Mouse models have been used to define the role of SK channels in diseases of the CNS, but the anatomical complexity of the mammalian nervous system and the existence of numerous redundant mechanisms to compensate for the loss of any protein limit the study of SK channels in this system. I therefore sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons have been mapped from their early developmental stages. KCNL-2 shares ~35% identity with the human SK2 and SK3 channels, with the greatest degree of conservation occurring in the six transmembrane domains, the pore motif and the calmodulin binding domain. The KCNL-2 gene was amplified from the WRM063DE08 fosmid and was fused to GFP at the amino and carboxy termini. Widefield and confocal fluorescence imaging of transgenic animals that expressed these constructs showed that KCNL-2 localizes to neurons of the nerve ring, pharyngeal nervous system, ventral nerve cord, dorsal cord, processes innervating the vulva, the ventral type-C neurons, mechanosensory neurons and in the lumbar ganglia. The complexity of the KCNL-2 gene was also demonstrated as the isoforms of KCNL-2 are differentially expressed due to varying promoter regions. Through phenotypic analysis of a KCNL-2 null strain and of transgenic lines that overexpress the channel, I demonstrated that KCNL-2 plays a role in the regulation of the rate of egg-laying. The KCNL-2 null strain was found to be mildly egg-laying defective while the transgenic lines that overexpress KCNL-2 showed a strong hyperactive egg-laying phenotype. I propose that overexpression of KCNL-2 hyperpolarizes unidentified neurons that inhibit egg-laying and subsequently causes a hyperactive egg-laying phenotype. With the ability to drive the expression of proteins in specific neuronal circuits, I propose that C. elegans is a sophisticated neurological model organism to study the biochemical, biophysical and physiological functions of SK channels.\ud \u