LOCALIZATION AND FUNCTIONALITY OF ION CHANNELS PRESENT IN THE DROSOPHILA MELANOGASTER AXON INTIAL SEGMENT
Open Access
- Author:
- Glatzer, Laura
- Graduate Program:
- Neuroscience
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- April 04, 2019
- Committee Members:
- Timothy J Jegla, Thesis Advisor/Co-Advisor
Melissa Rolls, Committee Member
Kevin Douglas Alloway, Committee Member - Keywords:
- voltage-gated potassium channels
axon initial segment
drosophila melanogaster
two-electrode voltage clamp
voltage-gated calcium channels
giant-ankyrin - Abstract:
- It is well established that vertebrate species use giant-Ankryin (Ank) as a major protein responsible for maintaining a barrier for the axon initial segment (AIS), which leads to the congregation of voltage-gated sodium, calcium, and potassium channels (Jenkins et al., 2015). These ion channels help with action potential (AP) generation and regulation in the AIS, which is known to generate APs and act as a barrier to keep somato-dendritic proteins out of the axon in vertebrates (Jones & Svitkina, 2016; Nelson & Jenkins, 2017). A recent study showed that a putative Ank2-dependent AIS is present in Drosophila sensory neurons, meaning a putative giant-ankyrin dependent AIS formed in a common bilaterian ancestor, earlier than previously assumed (T. Jegla et al., 2016). This thesis illustrates the localization of voltage-gated potassium channels (dmElk channels) and voltage-gated calcium channels (Cacophony channels) in the putative AIS and looks at the Ank2 and discs large (Dlg) scaffolding protein effect on localization of dmElk channels through fluorescent microscopy in Drosophila melanogaster. Using Drosophila melanogaster provides an in vivo model with powerful genetic tools to analyze the mechanisms of AIS formation and regulation, which may prove insightful to vertebrate processes. dmElk and the human ortholog Elk1 channels’ properties were analyzed through electrophysiology experiments using two-electrode voltage clamp. The experiments showed evidence that both channels activate at hyperpolarized voltages, suggesting they contribute to the regulation of subthreshold excitability. The mechanism behind the regulation of gating properties associated with Elk1 was looked at through the application of heme and zinc on the Elk1 channel. It was observed that heme causes a left shift of the channel’s gating properties allowing it to open at more hyperpolarized voltages. Meanwhile, zinc caused a right-shift suggesting a block of the channel, preventing it from opening until depolarized voltages are reached, by altering the extracellular charge clusters on S2 and S3 segments of the voltage sensors, preventing the stabilization of S4 segment that controls the level of block in the pore. The combination of zinc and heme caused the farthest right-shift resulting in the hypothesis that heme potentiates zinc, effectively sensitizing the channel to zinc, through the deprotonation of an external charge cluster. It is important to look at heme and zinc’s effects on activation of channels as tools to help us illustrate potential mechanisms behind the regulation of gating voltage-gated potassium channels. While heme and zinc are not directly at play in the physiological sense, understanding how they impact the channels may allow us to better understand what physiological conditions help regulate channel activation and ultimately action potential generation.