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

A Small Conductance Calcium-Activated K⁺ Channel in C. elegans, KCNL-2, Plays a Role in the Regulation of the Rate of Egg-Laying

In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying. © 2013 Chotoo et al

Phenotypic analysis of <i>kcnl-2</i>(<i>tm1885</i>) and KCNL-2(OE), a transgenic line that overexpresses <i>p</i>_{<i>kcnl-2</i>}<i>kcnl-2</i>(<i>taa2</i>)<i>::gfp</i> in the N2 background.

<p>A,C. Kaplan-Meier Survival Curves showing the longevities of N2 vs. <i>kcnl-2</i>(tm1885) (n=80; p=0.06, Logrank test) or N2 vs. KCNL-2(OE-2) (n=50; p=0.12, Logrank test). B,D. Brood size of N2 vs. <i>kcnl-2</i>(tm1885) (n≥17; p<0.001, Student’s <i>t</i> test) or N2 vs KCNL-2(OE-2) (n≥13; p=0.77, Student’s <i>t</i> test).</p

Confocal fluorescent images showing the expression pattern of the KCNL-2 promoter-GFP constructs.

<p>A–C. <i>p</i>_{<i>kcnl-2</i>(<i>atg1</i>)}<i>gfp</i> is expressed in neurons of the head and NR (A), the vulval muscles (B), and tail ganglia (C) (strain <i>vk1323</i>). D–F. <i>p</i>_{<i>kcnl-2</i>(<i>atg2</i>)}<i>gfp</i> is expressed in head neurons and NR (D); VNC, VC4 & VC5, and DC (E); and tail ganglia (F) (strain <i>vk1327</i>).</p

Structural analysis of KCNL-2 isoforms.

<p>A. The WRM063DE08 fosmid encodes six isoforms of KCNL-2, which vary in their amino and carboxy termini of the predicted protein structure. The exon structures show that there are two initiation sites that are 9.5 kb apart and two stop codons that are 68 bp apart. B) KCNL-2 isoforms have 37% identity with K _Ca2.2, while KCNL-2-aii and -b are 71% identical. The greatest degree of conservation occurs in the core of the channel where the S1-S6 transmembrane domains (underlined solid), the potassium-selective pore filter (underlined dotted), and the calmodulin binding domain (underlined dashed) are encoded. C) <i>kcnl-2</i>(tm1885), a 962 bp deletion, results in a premature stop codon before the first TMD of KCNL-2-aii and -b. D) Kyte-Doolittle hydropathy plot of the KCNL-2-a isoform showing seven domains that are highly hydrophobic, which represent the 6 TMDs and the pore motif (dashed line).</p

Rescue of the mild Egl phenotype shown by <i>kcnl-2</i>(<i>tm1885</i>) worms.

<p>A. <i>kcnl-2</i>(tm1885) animals retain a significantly greater number of eggs <i>in utero</i> relative to N2 animals (P<0.0001). The number of eggs <i>in utero</i> in heterozygotes (<i>kcnl-2</i>(-/+)) is significantly greater than that of the N2 animals (p=0.007), but significantly less than the average number of eggs found in <i>kcnl-2</i>(tm1885) animals (p=0.008). B. Transformation of <i>kcnl-2</i>(tm1885) with 1 ng/µl, 10 ng/µl or 90 ng/µl of <i>p</i>_{<i>kcnl-2</i>}<i>kcnl-2</i>(taa2)<i>::gfp</i> gives the transgenic lines KCNL-2(OE-3) (strain VK2220), KCNL-2(OE-4) (strain VK1004) and KCNL-2(OE-5) (strain VK1041), respectively. The average number of eggs retained <i>in utero</i> by KCNL-2(OE-3) was not significantly different from the N2 animals (P=0.8) but was significantly different from the <i>kcnl-2</i>(tm1885) organisms (p<0.0001). KCNL-2(OE-4) and KCNL-2(OE-5) retain fewer eggs <i>in utero</i> than N2 animals (p<0.005; p<0.0001, respectively) and <i>kcnl-2</i>(tm1885) animals (p<0.0001) (Student’s <i>t</i> test). C. Egg-staging assays show that the proportions of young eggs from KCNL-2(OE-2) (54.0±4.3%), KCNL-2(OE-4) (46.0±3.9%), and KCNL-2(OE-5) (46.6±6.7%) are significantly greater than those of N2 (7.2±1.3%) & <i>kcnl-2</i>(tm1885) worms (1.6±1.6%) (p<0.05; n=3, Kruskal–Wallis <i>H</i> test).</p

Overexpression of KCNL-2 in the N2 background causes a Hyperactive Egg-Laying Phenotype.

<p>A. An unlaid egg assay was carried out by dissolving the cuticle of adult worms in 1.8% sodium hypochlorite and shows that the number of eggs <i>in utero</i> in transgenic lines that overexpress KCNL-2 are significantly less than the number of eggs retained <i>in utero</i> in N2 and <i>kcnl-2</i>(tm1885) strains (p<0.001, Student’s <i>t</i> test), while <i>kcnl-2</i>(tm1885) has a significantly increased number of eggs retained <i>in utero</i> relative to N2 worms (p<0.001, Student’s <i>t</i> test). B) Representative images of worms from each strain. C) Egg-staging assays reveal that the proportion of young eggs from KCNL-2(OE-1) (strain VK1000) (54.82±7.65%) and KCNL-2(OE-2) (strain VK1065) (60.57±12.48%) was significantly greater than those of N2 worms (2.78±4.81%) (n=3, p<0.05; Kruskal–Wallis <i>H</i> test).</p