Biology subjects

Nimigean, C. M.

Publications and source records attributed to Nimigean, C. M..

7 recordsLinked to original sources

bioRxiv2026

Hydrophobic and lipid-mediated gating mechanism revealed by low-conductance MthK mutants

Large-conductance calcium (Ca2+)-activated potassium (K+) channels are involved in several essential cellular processes. This requires the ability to alternate between conductive and non-conductive states, yet the molecular mechanism of their gating remains incompletely understood. Bundle-crossing, selectivity filter, and hydrophobic gating are some of the mechanisms previously proposed for these channels, including the archaeal Ca2+-activated K+ channel MthK. Here, we took advantage of two MthK mutants that show dramatically-reduced conductance to gain insights into how the channels gate at the molecular level by combining cryo-electron microscopy, functional assays, and extensive molecular dynamics simulations. Functional and computational analyses reveal distinct mechanisms of reduced conductance between the two mutants. One mutant, where a glutamate residing at the intracellular mouth of the pore was replaced by an alanine (E92A) reduced ion density in the cavity and dramatically accelerated spontaneous channel closure. The second mutant, where an alanine residing in the channels aqueous cavity below the selectivity filter was replaced by a phenylalanine (A88F), introduced a hydrophobic constriction that partially dehydrates the cavity, imposing a barrier to ion permeation. The cryo-EM structures of these mutants, virtually identical to those of WT MthK, were used as models to investigate gating using molecular dynamics (MD) simulations. Long-timescale simulations captured complete open-to-closed transitions in both mutants, uncovering a dehydrated intermediate state and a tightly coupled lipid-entry mechanism that drove final pore closure. Our results show that hydrophobicity and lipid interactions are central determinants of MthK gating.

biophysics↗

bioRxiv2025

PIP2 Binding at Allosteric Site Blocks Activation in Human Rod CNG Channels

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a ubiquitous signaling lipid that regulates multiple ion channels. In human cyclic nucleotide-gated (CNG) channels, including the rod photoreceptor channel, PIP2 has been reported to exert inhibitory effects, but the underlying mechanism has remained unclear. Because this inhibition lowers the apparent cGMP sensitivity of rod CNG channels, it can play a key role in controlling the light sensitivity and dynamic range of rod photoreceptors. Here we report how PIP2 modulates the function of human CNGA1 channels, the major subunit of human rod photoreceptor CNG channels. Ensemble ion flux assays with liposome-reconstituted purified CNGA1 channels demonstrated robust inhibition by PIP2 via a reduction in apparent cGMP sensitivity, and single-channel recordings revealed PIP2 reduces the channels open probability without altering unitary conductance. To uncover the structural basis, we determined cryo-EM structures of CNGA1 in lipid nanodiscs under multiple ligand conditions. In PIP2-free conditions, closed, intermediate, and open conformations were observed, whereas in the presence of PIP2, the open state was absent. Density consistent with bound PIP2 was detected at inter-protomer grooves between the voltage-sensing and pore domains indicating that PIP2 binding stabilizes non-conductive conformations by sterically preventing C-linker elevation and outward movement of helix S6, conformational changes needed for pore dilation. Collectively, our results establish a structural mechanism for PIP2-mediated inhibition of rod CNG channels, define a mechanistic framework for phosphoinositide control of ligand-gated channels across the CNG superfamily, and provide an inhibitory allosteric binding site for future drug targeting in this channel family.

biophysics↗

bioRxiv2025

Mechanism of Lipid-Dependent Thermosensation in an Ion Channel

Temperature sensing is a fundamental biological process that allows organisms to detect and respond to environmental. Ion channels that gate in response to temperature changes, known as thermoreceptors, play key roles in this process. Although the phenomena affected by temperature in many thermoreceptors have been widely studied, the molecular mechanisms by which these proteins sense temperature remain controversial and poorly understood. Here, we investigated the molecular details of temperature sensing in a thermophilic bacterial ion channel, SthK from Spirochaeta thermophila, a homologue of mammalian hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels with well-characterized structure and function. We show that SthK is a cold-sensitive ion channel, displaying higher activity when temperatures are decreased below 30 {degrees}C. Intriguingly, the SthK cold sensitivity is highly dependent on the lipid composition, being sensitive in the presence of amine-containing lipids, and insensitive in the presence of anionic lipids. By combining cryo-EM structural analysis, mutagenesis, and functional assays, we demonstrated that a functionally important intersubunit salt bridge that was previously shown to be important in lipid modulation, acts as the temperature sensor in these channels. This salt bridge is state-dependent, stabilizes closed states, and needs to break for the channel to open. Lower temperatures that weaken salt bridge interactions thus favor channel opening. Lipid-binding at this location tune temperature sensitivity by modulating the salt bridge strength through their headgroup charge and size. Interesting to note, equivalent salt bridges are also found in other ion channels such as HCN channels and, notably, TRPM8, an established cold-sensitive thermoreceptor, suggesting that this mechanism could be conserved in other temperature-sensitive ion channels. Our findings highlight a finely tuned interplay between structural elements and membrane environment, suggesting that thermosensitivity can emerge from the cooperative effects of salt bridge energetics and lipid context.

biophysics↗

bioRxiv2020

Mechanistic insights into volatile anesthetic modulation of K2P channels

K2P potassium channels are known to be modulated by volatile anesthetic (VA) drugs and play important roles in clinically relevant effects that accompany general anesthesia. Here, we utilize a photoaffinity analog of the VA isoflurane to identify a VA binding site in the TREK1 K2P channel. The functional importance of the identified site was validated by mutagenesis and biochemical modification. Molecular dynamic simulations of TREK1 in the presence of VA found multiple neighboring residues on TREK1 TM2, TM3 and TM4 that contribute to anesthetic binding. The identified VA binding region contains residues that play roles in the mechanisms by which heat, mechanical stretch, and pharmacological modulators alter TREK1 channel activity and overlaps with positions found to modulate TASK K2P channel VA sensitivity. Our findings define molecular contacts that mediate VA binding to TREK1 channels and suggest a mechanistic basis to explain how K2P channels are modulated by VAs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Time-resolved cryoEM using Spotiton

We present an approach for preparing cryoEM grids to study short-lived molecular states. Using piezo electric dispensing, two independent streams of ~50 pL sample drops are deposited within 10 ms of each other onto a nanowire EM grid surface, and the mixing reaction stops when the grid is vitrified in liquid ethane, on the order of ~100 ms later. We demonstrate the utility of this approach for four biological systems where short-lived states are of high interest.

biophysics↗

bioRxiv2019

Direct Binding of Phosphatidylglycerol at Specific Sites Modulates Desensitization of a Pentameric Ligand-Gated Ion Channel

Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, the L240A mutant known to decrease ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites.

biophysics↗

bioRxiv2018

Cryo-EM structures reveal bilayer remodeling during Ca2+ activation of a TMEM16 scramblase

The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several members of the TMEM16 family have been identified as Ca2+-activated scramblases but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs, revealing large Ca2+-dependent conformational changes of the protein as well as significant, function dependent membrane reorganization.

biochemistry↗