Gucan "Gabriel" Dai, Ph.D.
Assistant Professor
Biophysical and structural mechanisms of ion channels, principles of bioelectricity, and the biochemistry of excitable membranes.
Research Interests
Ion channels form ion-permeable transmembrane pores that govern the so-called “nerve spikes or action potentials” and rapidly transmit electrical signals in excitable cells. They can be viewed as specialized enzymes that catalyze the fast and selective diffusion of ions, like sodium and calcium ions across the cell membrane. These macromolecules are crucial for the electrical activities that are responsible for many essential physiological processes; for example: neurotransmitter release, synaptic transmission, and cardiac pacemaking.
The Dai lab combines cutting-edge biophysical and biochemical techniques, including patch-clamp fluorometry, spectroscopic FRET, protein engineering, fluorescent noncanonical amino acids, and high-resolution nanoscopy, to understand how ion channels are controlled by membrane voltages, the binding of intracellular ligands, and the rapid dynamics of cellular lipids. These mechanisms are important for major diseases, including neuropathic pain, epilepsy, Alzheimer's disease, and cardiac arrhythmia.
Projects in the lab include:
- Voltage-sensing and regulatory mechanisms of pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. The HCN channel governs cardiac pacemaking and plays a role in controlling the firing of nociceptive neurons. To understand how HCN channels are implicated in the cellular and structural basis of pain sensation, experimental plans will focus on the structural mechanisms of human HCN channels and on the channel modulation that is related to the neuropathic pain.
- Understanding how ion channels depend on their lipid environment, especially the dynamics of key signaling phosphoinositides and the lipid-raft microdomains.
- Future interests of the lab include the biophysics of neuronal voltage-gated potassium channels as well as the cyclic nucleotide-gated channels in human retinal photoreceptors.
Recent Publications
Sequential membrane remodeling by cholesterol distinctly modulates HCN channels in naïve and neuropathic DRG neurons
Sequential membrane remodeling by cholesterol distinctly modulates HCN channels in naïve and neuropathic DRG neurons
Cholesterol, abundantly present in distinct plasma membrane pools, is a critical modulator of ion channel function, including hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that regulate the excitability of dorsal root ganglion (DRG) nociceptor neurons. Depletion of membrane cholesterol potentiated HCN channel opening and accelerated activation kinetics, whereas cholesterol supplementation reduced channel opening and slowed activation kinetics. However, the relative contributions of cholesterol that organizes ordered membrane domains (OMDs) versus freely accessible cholesterol pools to HCN channel modulation remain unknown. Using fluorescence lifetime imaging microscopy, FRET and fluorescence anisotropy techniques, we examined how supplementing cholesterol alters plasma membrane properties and HCN gating in nociceptor DRG neurons. We uncovered a process of sequential, stepwise membrane remodeling: an initial phase with OMD expansion and a rapid rise in free cholesterol, followed by continued accumulation of free cholesterol without further OMD expansion. Notably, the slope factor of the HCN G-V relationship is sensitive to OMD expansion but remains unaffected by changes in free cholesterol. Other gating parameters, including open probability and activation kinetics, were affected by elevating free cholesterol. In a rat model of nerve injury, where DRG neurons exhibit reduced free cholesterol levels and smaller OMDs, HCN channel modulation by cholesterol involves contributions from both OMD expansion and free cholesterol accumulation. In contrast, in naïve DRG neurons-characterized by high cholesterol and large OMDs-modulation occurs mostly via increased free cholesterol. These findings provide mechanistic insights into cholesterol-dependent modulation of ion channels and its role in neuropathic pain.
Voltage sensor conformations induced by LQTS-associated mutations in hERG potassium channels
Voltage sensor conformations induced by LQTS-associated mutations in hERG potassium channels
Voltage sensors are essential for electromechanical coupling in hERG K channels, critical to cardiac rhythm. These sensors respond to membrane potential changes by moving within the transmembrane electric field. Mutations in hERG voltage-sensing arginines, associated with Long-QT syndrome, alter channel gating, though underlying mechanisms remain unclear. Using live-cell fluorescence lifetime imaging microscopy, transition metal FRET, an improved dual stop-codon-mediated strategy for noncanonical amino-acid incorporation, and molecular dynamics simulations, we identify intermediate voltage-sensor conformations induced by neutralizing key arginines in the charge transfer center. Phasor plot analysis of lifetime data reveals multiple voltage-dependent FRET states in these mutants, in contrast to the single high-FRET state observed in controls. These intermediate FRET states reflect distinct conformations of the voltage sensor, corresponding to predicted structures of voltage sensors in molecular dynamics simulations. This study provides insights into cardiac channelopathies, highlighting a structural mechanism that impairs voltage sensing in cardiac arrhythmias.
Membrane lipid nanodomains modulate HCN pacemaker channels in nociceptor DRG neurons
Membrane lipid nanodomains modulate HCN pacemaker channels in nociceptor DRG neurons
Cell membranes consist of heterogeneous lipid nanodomains that influence key cellular processes. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we find that the dimension of cholesterol-enriched ordered membrane domains (OMD) varies considerably, depending on specific cell types. Particularly, nociceptor dorsal root ganglion (DRG) neurons exhibit large OMDs. Disruption of OMDs potentiated action potential firing in nociceptor DRG neurons and facilitated the opening of native hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels. This increased neuronal firing is partially due to an increased open probability and altered gating kinetics of HCN channels. The gating effect on HCN channels is likely due to a direct modulation of their voltage sensors by OMDs. In animal models of neuropathic pain, we observe reduced OMD size and a loss of HCN channel localization within OMDs. Additionally, cholesterol supplementation inhibited HCN channels and reduced neuronal hyperexcitability in pain models. These findings suggest that disturbances in lipid nanodomains play a critical role in regulating HCN channels within nociceptor DRG neurons, influencing pain modulation.
Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization
Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization
Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated ion channels to transmembrane potential within distinct membrane compartments. By leveraging fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), we visualized the localization of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in membrane domains. HCN4 exhibits a greater propensity for incorporation into ordered lipid domains compared to HCN1. To investigate the conformational changes of the S4 helix voltage sensor of HCN channels, we used dual stop-codon suppression to incorporate different noncanonical amino acids, orthogonal click chemistry for site-specific fluorescence labeling, and transition metal FLIM-FRET. Remarkably, altered FRET levels were observed between VSD sites within HCN channels upon disruption of membrane domains. We propose that the voltage-sensor rearrangements, directly influenced by membrane lipid domains, can explain the heightened activity of pacemaker HCN channels when localized in cholesterol-poor, disordered lipid domains, leading to membrane hyperexcitability and diseases.
Signaling by Ion Channels: Pathways, Dynamics and Channelopathies
Signaling by Ion Channels: Pathways, Dynamics and Channelopathies
Charged ions and ion channels play a critical role in regulating the electrical activities of excitable cells. This review discusses the principles of ion channel regulation in the time domain, as well as the diseases that can arise from channel dysfunction and disturbances in ionic balance. Ion channel signaling is a dynamic process that is essential for various physiological functions, including pain sensation, motor control, and the body’s response to stress, such as fight-or-flight response.
