Biology subjects

Stephens, C. A.

Publications and source records attributed to Stephens, C. A..

3 recordsLinked to original sources

bioRxiv2026

Conformational changes upon pore blocker removal reveal conductive states of TMEM16A

TMEM16A is a Ca2+-activated anion channel that provides direct electrical feedback to the plasma membrane in response to intracellular Ca2+. Its conductive state remains unresolved, leaving questions about gating, Cl- permeation, and modulation by Ca2+, depolarization, and lipids. To investigate the open state, we performed molecular dynamics simulations of TMEM16A bound to the putative open-state blocker 1PBC. After inhibitor removal, the putative, pore-lining helix TM4 developed kinks at two sites: an upper site that opens the pore for Cl- permeation, and a deeper site causing constriction. A conserved hydrophobic network between TM3 and TM4 persisted in most open structures but separated during extreme dilation, allowing lipids to transiently block the pore. Patch-clamp recordings indicated that the intact network promotes activation. Further simulations yielded >60 Cl- permeation events and a single-channel conductance matching experiments. Additional electrostatic and kinetic modeling indicated that TMEM16As transition from outward-rectification to Ohmic conductance with increasing Ca2+ results from a weak voltage dependence of Ca2+ binding, which act cooperatively to open the pore. HIGHLIGHTSO_LIRemoval of the inhibitor 1PBC from TMEM16A induces spontaneous transitions to ion conductive and non-conductive states via bending in TM4 at two different locations. C_LIO_LISimulations suggest that the open state is stabilized by a small hydrophobic network between TM3 and 4 and disrupting this network biases channel closure in electrophysiological recordings. C_LIO_LIA kinetic model of conduction based on energetics from the all-atom MD simulations coupled to continuum calculations gives a linear current-voltage curve consistent with the fully open conformation and removal of 1 Ca2+ switches to an outwardly rectifying state via electrostatic influence on the Cl- energy profile. However, the rectification is too weak to match experiment, but a cooperative model of Ca2+ binding with weak voltage-dependence does match experiment. C_LI

biophysics↗

bioRxiv2024

Robust lipid scrambling by TMEM16 proteins requires an open groove and thin membrane

Biological membranes are complex and dynamic structures with different populations of lipids in their inner and outer leaflets. The Ca2+-activated TMEM16 family of membrane proteins plays an important role in collapsing this asymmetric lipid distribution by spontaneously, and bidirectionally, scrambling phospholipids between the two leaflets, which can initiate signaling and alter the physical properties of the membrane. While evidence shows that lipid scrambling can occur via an open hydrophilic pathway ("groove") that spans the membrane, it remains unclear if all family members facilitate lipid movement in this manner. Here we present a comprehensive computational study of lipid scrambling by all TMEM16 members with experimentally solved structures. We performed coarse-grained molecular dynamics (MD) simulations of 27 structures from five different family members solved under activating and non-activating conditions, and we captured over 700 scrambling events in aggregate. This enabled us to directly compare scrambling rates, mechanisms, and protein-lipid interactions for fungal and mammalian TMEM16s, in both open (Ca2+-bound) and closed (Ca2+-free) conformations with statistical rigor. We show that all TMEM16 structures thin the membrane and that the majority of scrambling (>90%) occurs at the groove only when TM4 and TM6 have sufficiently separated. Surprisingly, we also observed 60 scrambling events that occurred outside the canonical groove, over 90% of which took place at the dimer-dimer interface in mammalian TMEM16s. This new site suggests an alternative mechanism for lipid scrambling in the absence of an open groove. Impact StatementThe majority of TMEM16 lipid scrambling occurs in the open groove associated with Ca2+-activation, but limited scrambling also occurs in the dimer interface independent of Ca2+.

biophysics↗

bioRxiv2020

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of -synuclein, a dynamic protein linked to Parkinsons disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of -synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to -synuclein biology and pathobiology. We found that the conformation of -synuclein previously shown to drive yeast toxicity--an extended, membrane-bound helix--is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress -synuclein toxicity. Moreover, we find that sequence determinants of -synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that -synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

biochemistry↗