Biology subjects

Handlin, L. J.

Publications and source records attributed to Handlin, L. J..

5 recordsLinked to original sources

bioRxiv2025

RhoA activation promotes ordered membrane domain coalescence and suppresses neuronal excitability

The formation of ordered proteolipid membrane domains (OMDs) within the plasma membrane has emerged as a fundamental process that modifies membrane function, particularly in response to cell stresses that promote pathological states. Here, we identify a previously unrecognized role for the small GTPase RhoA to promote the coalescence of OMDs, thereby linking cytoskeletal remodeling and membrane mechanics to OMD formation. Pharmacological and optogenetic manipulation of RhoA rapidly altered OMD dimensions in both human cell lines and dorsal root ganglion (DRG) nociceptors. The RhoA-dependent OMD expansion required actin remodeling, changes in membrane mechanical state, and protein palmitoylation. Functionally, RhoA inhibition increased action potential firing and potentiated HCN channel activity in DRG neurons. Conversely, in a spared nerve injury model characterized by altered membrane mechanics, reduced OMD size, and hyperexcitability, RhoA activation enlarged OMDs, suppressed HCN channel activity, and reduced firing. These findings highlight alterations in plasma membrane physical properties, including changes in OMD organization and membrane tension, as key features of neuropathic stress. RhoA/ROCK-driven OMD remodeling may serve as a compensatory membrane adaptation that counteracts neuropathic hyperexcitability.

biophysics↗

bioRxiv2025

Cholesterol Inhibits HCN Channels through Dual Mechanisms in Neuropathic Pain

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 and Forster resonance energy transfer (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 conductance- voltage 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 naive DRG neurons--characterized by high cholesterol and large OMDs--modulation occurs mostly via increased free cholesterol. These findings deepen our understanding of cholesterols role in modulating ion channels and contributing to neuropathic pain.

neuroscience↗

bioRxiv2024

Phasor FLIM-FRET Analysis of Voltage Sensor Conformational Heterogeneity 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 (FLIM), transition metal FRET (tmFRET), an improved dual stop-codon-mediated strategy for noncanonical amino-acid incorporation, and molecular dynamics (MD) simulations, we identified intermediate voltage-sensor conformations induced by neutralizing key arginines in the charge transfer center. Phasor plot analysis of FLIM data revealed 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 MD simulations. This study provides novel insights into cardiac channelopathies, highlighting a structural mechanism that impairs voltage sensing in cardiac arrhythmias.

biophysics↗

bioRxiv2023

Disruption of Ordered Membrane Domains as a Mechanism Underlying Neuropathic Pain

Cell membranes consist of heterogeneous lipid nanodomains that influence key cellular processes. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we found 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 was likely due to a direct modulation of their voltage sensors by OMDs. In animal models of neuropathic pain, we observed 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.

biophysics↗

bioRxiv2023

DIRECT REGULATION OF THE VOLTAGE-SENSING DOMAIN 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 Forster 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.

physiology↗