Evolutionary Mechanism Governing Protein Multimerization in Cnidarian Shak6 and ShakR2
Restricted (Penn State Only)
- Author:
- Schreiber, Madelynn
- Area of Honors:
- Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Timothy J Jegla, Thesis Supervisor
Bernhard Luscher, Thesis Honors Advisor - Keywords:
- Electrophysiology
Cnidarians
Evolution
Kv Channels
Neuroscience
Shaker Channels - Abstract:
- Transmembrane proteins that are selectively permeable to particular ions and triggered by membrane depolarization or hyperpolarization are known as voltage-gated ion channels. These channels are essential for producing electrical impulses across cell membranes. In order to stop cellular signaling, voltage-gated potassium (Kv) channels allow potassium ions to cross the membrane down the electrochemical gradient which leads to membrane repolarization following depolarization. Voltage-gated K+ channels, crucial for neuronal and muscular excitation control, vary in number across bilaterian species, with humans and mice possessing up to 40 different genes. Among the primary gene families encoding these channels, the Shaker family is of particular interest, having been characterized initially in Drosophila and showing diversification in cnidarians, offering insight into their evolutionary origins and unique phenotypes. Structurally, Kv channels consist of α-subunits forming a tetramer, with distinct domains including voltage sensors (S1-S4) and pore regions (S5-S6) housing the K+ selectivity filter. The T1 domain, a family-specific cytoplasmic region located at the N terminus, and the voltage-gated K+ channel core make up the subunit structure that sets Shaker family channels apart from the other K+ channel families. Different Kv channel subunits exhibit adaptable expression patterns and biophysical characteristics, forming homo- or heterotetrameric channels within four subfamilies (Kv1-4). The T1 domain controls tetramerization by preventing interactions between incompatible subunits, thus influencing assembly specificity. Interactions between subunits involve polar interactions, with highly preserved amino acids encoding subfamily-specific assembly characteristics. Across Kv1-4 subfamilies, the T1 core remains similar, emphasizing the importance of subfamily-specific interactions for assembly specificity. While Kv1-4 subunits typically form functional homotetramers, regulatory subunits, found in mammals and independently evolved in cnidarians, are self-incompatible but can form heteromeric channels within the same subfamily. Multiple regulatory subunits have evolved in both chordates and cnidarians, suggesting convergent evolution of assembly mechanisms within the Shaker subfamily. We hypothesize that a prerequisite for heteromeric-dependent assembly in Kv R-subunits must have evolved by a loss-of-function mutation in the T1 assembly domain that specifically prevents self-recognition and homomer formation. Our hypothesis posits that introducing ancestral R2/R3 mutations into Shak6 disrupts channel assembly, altering activation rate, inactivation rate, and voltage-dependence. Shak6, chosen as the alpha subunit of interest due to its monophyletic clade with regulatory subunits present in stem cnidarians, allowed examination of the origin of a regulatory phenotype within an alpha-subunit clade. The mutations, identified through molecular dynamic simulations and Ancestral Sequence Reconstruction (ASR), were introduced into Shak6 subunits to investigate the evolution of ShakR2 regulatory subunits within the assembly phenotype. Starting with alpha subunits was essential as they form functional homomeric channels, unlike regulatory subunits, which lack a functional gating domain. Analysis of the mutants revealed no statistically significant difference in average peak current compared to wild type Shak6, suggesting that they maintain function similar to the wild type channels and do not alter channel assembly or gating. However, differences in example current traces hint at a possible role for these mutations in channel assembly. The limited knowledge of Kv channels in ancestral metazoans impedes our understanding of electrical signaling evolution, underscoring the importance of comparing channel structure, function, and regulation between species to elucidate shared ancestry and evolutionary pathways. Although this study faces limitations due to variance in current sizes and the need for further electrophysiological trials, investigating the impact of the T1 domain on channel assembly through truncation experiments could shed light on its role and evolutionary significance. Despite evidence suggesting robust channel assembly without a functional T1 domain in vitro, further research is needed to understand its role in vivo, and exploring additional regulatory subunit clades in cnidarians could provide valuable insights into the evolution of Kv channels.