Investigating the Evolution of Inward and Outward Rectifying Phenotypes in Plant Voltage Gated Potassium Channels
Restricted (Penn State Only)
- Author:
- Puckerine, Mikaili
- 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
plant
algae
ion channels - Abstract:
- This thesis combines an electrophysiology and evolutionary approach to understanding the different gating phenotypes of plant voltage gated potassium (plant Kv) channels. Voltage gated potassium channels in flowering plants belong to a single extended gene family that can be divided into two groups: those that are activated by depolarization (outward rectifying channels) and those that are activated by hyperpolarization (inward rectifying channels). Using a phylogenetic comparison of channel amino acid sequences from diverse plant species, we were able to trace the molecular lineage of each channel’s gating type back to an origin in the algae family. However, the functional phenotypes of these channel outside the flowering plant are unknown. We wanted to express channels from more primitive plants to determine if these clades have had distinct voltage gating phenotypes throughout the evolutionary history of plants. We picked Streptophyte algae to test first because it is the earliest diverging species clade in which we can clearly identify inward and outward channel data collected in the phylogenetic tree. Within the Streptophyte algae species clade, we picked the Spirogloea muscicola algae species because it contains both inward and outward ion channels with a relatively complete genomic dataset that can be tested. We chose the outward potassium channel Spirogloea muscicola_SM000037S13469 (SmusKvout1) and the inward potassium channel Spirogloea muscicola_SM000099S25200 (SmusKvin1) to functionally characterize. Complementary DNAs for both channels were cloned into plasmids, transcribed into RNA, synthesized into RNA, injected into oocytes, and then tested for functional expression using two electrode voltage clamp (TEVC) recordings. Various tests were completed to assess functional expression including changes in the external potassium concentration, pH, introducing divalent cations, and co-expression and testing for heteromeric channel formation with functional channels from the flowering plants. However, despite testing in all these different conditions, both SmusKvin1 and SmusKvout1 showed no functional expression. Hypothetically, these two channels could be regulatory subunits that are unable to conduct current by themselves. In terms of future directions, we would also want to explore co-injections with other subunits from Spirogloea muscicola species to test for the formation of heteromeric tetramers that can conduct current. We also want to pick other clades in plant Kv like the Liverworts or Ferns that are close to flowering plant clade to try and functionally express. If these channels can conduct current by themselves, it gives us more information on the phenotype in early land plants.