Publication Date




Embargo Period


Degree Type


Degree Name

Doctor of Philosophy (PHD)


Neuroscience (Medicine)

Date of Defense


First Committee Member

Laura Bianchi

Second Committee Member

Gerhard Dahl

Third Committee Member

Peter H. Larsson

Fourth Committee Member

Julia Dallman

Fifth Committee Member

Samuel M. Young


Ion channels of the DEG/ENaC family can induce neurodegeneration under conditions in which they become hyperactivated. The C. elegans DEG/ENaC channel MEC-4(d) encodes a mutant channel with a substitution in the pore domain that causes swelling and death of the six touch neurons in which it is expressed. Dominant mutations in homolog channel UNC-8 result in uncoordinated movement. Here we show that this unc-8(d) movement defect is correlated with the selective death of cholinergic motor neurons in the ventral nerve cord, a milder toxicity compared with MEC-4(d). UNC-8(G387E), denoted UNC-8(d), and UNC-8(A586T) mutants encode hyperactivated channels that are strongly inhibited by extracellular calcium and magnesium in Xenopus oocytes. Reduction of extracellular divalent cations exacerbates UNC-8(d) toxicity in oocytes. We suggest that inhibition by extracellular divalent cations limits UNC-8 toxicity and may contribute to the selective death of neurons that express UNC-8 in vivo (Chapter 3). It was previously shown that neurons expressing hyperactive DEG/ENaC channels die by necrosis mediated by intracellular Ca2+ overload. Hyperactive mammalian ASIC1a and C. elegans MEC-4(d) conduct both Na+ and Ca2+ raising the possibility that direct Ca2+ influx through these channels contributes to the intracellular Ca2+ overload. However, we showed that UNC-8(d) is not Ca2+ permeable but still causes neuronal death suggesting that Na+ influx is sufficient to induce cell death. MEC-4(d) and UNC-8(d) differ not only in Ca2+ permeability but also in current amplitude, UNC-8 being strongly blocked by physiologic Ca2+ concentration. Given that these two channels show a striking difference in toxicity in vivo, we asked what is the contribution of Na+ conductance and Ca2+permeability to cell death. To address this question we built an UNC-8/ MEC-4 chimeric channel that retains the calcium permeability of MEC-4 and characterized its properties in oocytes. Our data support the hypothesis that for Ca2+ permeable DEG/ENaC channels, such as MEC-4, both Ca2+ permeability and Na+ conductance contribute to toxicity. However, for Ca2+ impermeable DEG/ENaCs, such as UNC-8, constitutive Na+ conductance is sufficient to induce toxicity and this effect is enhanced as current amplitude increases (Chapter 4). UNC-8(d) is blocked by extracellular Ca2+ in the non-physiologic micromolar range but causes neuronal death. This suggests that homolog DEG/ENaC subunits and accessory proteins that are co-expressed with UNC-8 in cholinergic motorneurons in vivo may modulate its Ca2+ sensitivity. I found that none of the selected proteins changed UNC-8 Ca2+ sensitivity when co-expressed with UNC-8 in oocytes (Chapter 5). Reduction of extracellular divalent cations exacerbates UNC-8(d) toxicity in oocytes suggesting that inhibition by extracellular divalent cations limits UNC-8 toxicity and may contribute to the selective death of neurons that express UNC-8 in vivo. To test this hypothesis I proposed to investigate a transgenic C. elegans with mutant UNC-8(d) channel in which the calcium binding site has been altered, to see if removing the calcium block will change the level of channel toxicity in vivo. Using a chimeric approach we swapped extracellular domains of UNC-8 with the corresponding domains of the less calcium sensitive MEC-4. We find that residues in the extracellular “finger” domain are responsible for UNC-8(d) divalent cation sensitivity. Furthermore, our results show for the first time that residues in this extracellular “finger” domain are involved in the interaction of MEC-4 channel with accessory protein MEC-6 (Chapter 5). This work furthers refines the contribution of different channel properties to cellular toxicity induced by hyperactive DEG/ENaC channels and expands on previous structure-function studies by establishing properties and functions of DEG/ENaC channels specific domains.


DEG/ENaC channels, neurotoxicity, calcium block, calcium permeability