Date of Degree
MS (Master of Science)
Daniel F. Eberl
Ion pumps establish homeostasis across the membranes of living cells. Hearing is a mechanotransduction event that takes place in a closed compartment containing a fluid high in K+ concentrations. In Drosophila melanogaster, this closed compartment is formed by a scolopale cell that wraps around the dendrite of sensory neurons. The receptor lymph is maintained by the scolopale cell. The lumenal membrane of the scolopale cell is the wall of the compartment containing the receptor lymph, the scolopale space. The ablumenal membrane of the scolopale cell creates the border of the scolopidium.
The Na/K pump is located on the ablumenal membrane of the scolopale cell, bringing K+ into the scolopale cell cytoplasm and extruding K electrogenically (Roy et al, 2013). We explored other primary and secondary ion pumps that are involved in creating a K+-rich lumen in the Malpighian tubule (Day et al, 2008; Rodan et al, 2012). We used RNAi technology to knockdown one gene at a time and electrophysiology to measure a sound evoked potential (SEP) that reflects the fly’s ability to hear.
We found that knocking down V-ATPase, a proton pump, subunits involved in proton extrusion significantly reduces the SEP of knockdown flies. The involvement of cation chloride cotransporters (CCCs) and cation proton antiporter (CPAs), both secondary ion pumps that use the gradients created by the Na/K pump and V-ATPase respectively to pump other ions up their gradient, is less clear. We found that knocking down Nhe3, a CPA, significantly reduced the SEP when knocked down in the scolopale cell, suggesting it as a partner to the V-ATPase. Knocking down CG31547, a CCC, statistically increased the SEP, possibly a type1 statistical error.
The organ the fruitfly uses to hear contains a fluid called the receptor lymph. Cells use ion pumps to maintain ion concentrations in fluid-filled compartments. Here, cells surrounding the receptor lymph were genetically manipulated so that they do not produce each of three kinds of ion pumps. This will let us test if they are important in fly hearing. These pumps are also important for human hearing so understanding how these pumps work may help understand and perhaps prevent hearing loss.
We explored the proton pump, which acidifies specialized compartments, and found it to be important in mediating hearing. The second type of pump, the cation proton antiporters (CPAs) use the energy provided by the proton pump to produce a compartment concentrated in an ion key for fruitfly (and human) hearing. And the third kind of pump was called the cation chloride cotransporters (CCCs). We found that flies lacking Nhe3, a kind of CPA, in the structures responsible for making the receptor lymph were not able to hear as well. In addition, Nha2, another type of CPA, is located in the specialized cells maintaining the receptor lymph; this candidate we could not test functionally.
In conclusion, we provided evidence that the proton pump may work with Nhe3 and Nha2 to maintain proper ion concentrations in the receptor lymph, but more work needs to be done to understand how these three pumps interact.
publicabstract, Cation Chloride Cotransporters, Cation Proton Antiporters, Hearing, Mechanosensation, Proton Pump, Scolopidium
x, 49 pages
Includes bibliographical references (pages 43-49).
Copyright 2015 Betul Zora