ORCID
https://orcid.org/0000-0001-9116-1763
Date of Award
5-8-2025
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Key physiological processes such as pacemaking in the heart, electrical signaling in the brain, photoreception in the eye, olfaction in olfactory sensory neurons, and stomata opening in plant leaves involve channels from the cyclic nucleotide binding domain (CNBD) ion channel family. CNBD channels are found throughout the tree of life, and for those with cryo-EM structures, show striking similarity in their architecture despite exhibiting diversity in gating polarity (i.e. channels that activate upon membrane hyperpolarization, depolarization, or are voltage-insensitive). Single mutations in CNBD channels can not only reverse gating polarity, but can also create channels that activate upon both membrane hyperpolarization and depolarization. To our knowledge, this plasticity in gating polarity is unique to the CNBD family within the superfamily of voltage-gated ion channels. In this thesis, I discuss the functional and molecular evolutionary approaches I used to determine the interactions between multiple structural elements involved in gating polarity. Voltage-gated members of the CNBD family are found primarily in three subfamilies: hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, ether-à-go-go (EAG) channels, and Plant Voltage-Gated K+ (Plant VG K+) channels. Prior structural and functional studies show that different interactions between the pore domain and the voltage-sensing domain contribute to the diversity of gating polarity observed in these subfamilies. However, chimeragenesis studies reveal that other structural domains may also be involved in channel gating. In Chapter 2, I assess the impact of the C-terminal domain on gating polarity by swapping the C-terminus from members of the HCN, EAG, and Plant VG K+ channel families into a chimeric background. I then proceeded to perform alanine-scanning mutagenesis of the C-terminus to identify specific residues with the greatest contribution to gating polarity. In Chapter 3, I generated a state equilibrium model to interpret the gating scheme for all CNBD channels. To empirically test and validate the function of each subfamily, I used electrophysiology to characterize select extant CNBD channels in Chapter 4. These select extant channel species are least molecularly divergent to their respective subfamily’s ancestral channel, thus, giving us insight into residues and interactions that remain through evolutionary time key to channel gating polarity. Lastly, Chapter 5 is a discussion of the work presented in this thesis and future directions for molecular evolutionary and functional analyses of gating polarity in CNBD channels. Taken altogether, this body of work explores the molecular determinants for gating polarity through a functional and molecular evolutionary lens.
Language
English (en)
Chair and Committee
Baron Chanda
Committee Members
Alex Holehouse; Christopher Lingle; Janice Robertson; Michael Landis
Recommended Citation
Lin, Jenna L., "Mechanisms of Voltage Gating in the Cyclic Nucleotide Binding Domain Channel Family" (2025). Arts & Sciences Theses and Dissertations. 3526.
https://openscholarship.wustl.edu/art_sci_etds/3526