Biology subjects

Bankston, J. R.

Publications and source records attributed to Bankston, J. R..

9 recordsLinked to original sources

bioRxiv2026

The proximal N-terminus of IRAG is required for potentiation of HCN4 channels

The inositol triphosphate-associated, ER transmembrane proteins IRAG and LRMP are isoform specific regulators of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, while IRAG mimics the effect of cAMP on the channel. We previously showed that regulation by LRMP requires both the N-terminus of HCN4 and a unique orientation of the HCN4 cAMP transduction center, which is comprised of the N-terminal HCN domain, the C-linker, and the S4-S5 linker. However, it remains unknown if the homologous IRAG requires similar structural features to mimic cAMP-dependent potentiation, or if the site and mechanism of action are different between the two regulators. Using patch clamp electrophysiology, we determined that the initial 43 amino acids of IRAG are necessary and sufficient to confer regulation of HCN4. Similar to LRMP, IRAG also requires a portion of the N-terminus of HCN4 to confer its regulatory effects. Also similar to LRMP, two point mutations in the C-linker region, which are the only sequence differences in that region between HCN4 and the other HCN isoforms, were able to eliminate the effect of IRAG suggesting the unique orientation of the cAMP transduction center in HCN4 is likely important for IRAG function. Taken together, these findings suggest a model whereby IRAG and LRMP interact with the channel in similar regions, although potentially in unique ways, and act on the cAMP transduction center with LRMP inhibiting the coupling of this region to gating and IRAG strengthening it. SUMMARYThe ER transmembrane protein IRAG binds to and potentiates HCN4 channels. This study demonstrates that IRAG regulation of HCN4 requires only the first 43 amino acids of IRAG and involves contributions from the N-terminus and cAMP transduction center of HCN4.

biophysics↗

bioRxiv2026

PUFA modulation of ASIC3 involves both specific and lipid solvent-like interactions

AbstractInflammatory mediators including polyunsaturated fatty acids (PUFAs) potentiate ASIC3 by altering channel gating, yet the molecular basis for their interactions remains poorly defined. Using all-atom MD simulations and electrophysiology, we show that DHA accumulates around ASIC3 through interactions with a membrane-facing electropositive region along the outer leaflet of TM1. In the open state, a more buried binding site for the lipid is exposed that allows the carboxylate head group to bind a critical arginine along with nearby polar residues. Mutation of this site prevents the slowing of channel desensitization but only reduces the effect PUFAs have on the pH dependence of activation. The same mutations do not prevent regulation by other lipid classes like N-acyl amino acids or lysophosphatidyl choline. Our results provide the first detailed description of a PUFA binding site on ASICs, offering new insights into lipid modulation and potential strategies for developing novel treatments for inflammatory pain.

biophysics↗

bioRxiv2024

Elucidating the Differential Impacts of Equivalent Gating-Charge Mutations in Voltage-Gated Sodium Channels

Voltage-gated sodium (Nav) channels are pivotal for cellular signaling and mutations in Nav channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function (GoF), loss-of-function (LoF) effects, or both. However, the mechanism behind this functional divergence of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino-acid substitutions at equivalent gating-charge positions in VSDI and VSDII of the cardiac sodium channel Nav1.5 show differential gating-property impacts in electrophysiology measurements. We conducted 120 {micro}s molecular dynamics (MD) simulations on wild-type and six mutants to elucidate the structural basis of their differential impacts. Our s-scale MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis elucidated how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study represents a significant leap forward in understanding structure- function relationships in Nav channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel-related disorders.

biophysics↗

bioRxiv2024

Molecular Insights into Single Chain Lipid Modulation of Acid-Sensing Ion Channel 3

Polyunsaturated fatty acids (PUFAs) and their analogs play a significant role in modulating the activity of diverse ion channels, and recent studies show that these lipids potentiate acid-sensing ion channels (ASICs), leading to increased activity. The potentiation of the channel stems from multiple gating changes, but the exact mechanism of these effects remains uncertain. We posit a mechanistic explanation for one of these changes in channel function, the increase in the maximal current, by applying a combination of electrophysiology and all-atom molecular dynamics simulations on the open-state hASIC3. Microsecond-scale simulations were performed on open-state hASIC3 in the absence and presence of a PUFA, docosahexaenoic acid (DHA), and a PUFA analog, N-arachidonyl glycine (AG). Intriguingly, our simulations in the absence of PUFA or PUFA analogs reveal that a tail from the membrane phospholipid POPC inserts itself into the pore of the channel through lateral fenestrations on the sides of the transmembrane segments, obstructing ion permeation through the channel. The binding of either DHA or AG prevented POPC from accessing the pore in our simulations, relieving the block of ionic conduction by phospholipids. Finally, we use the single-channel recording to show that DHA increases the amplitude of the single-channel currents in ASIC3, which is consistent with our hypothesis that PUFAs relieve the pore block of the channel induced by POPCs. Together, these findings offer a potential mechanistic explanation of how PUFAs modulate ASIC maximal current, revealing a novel mechanism of action for PUFA-induced modulation of ion channels.

biophysics↗

bioRxiv2023

LRMP inhibits the cAMP-dependence of HCN4 channels by disrupting intramolecular signal transduction

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4 but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here we identify the domains of LRMP essential for regulation. We show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating. And we demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Forster resonance energy transfer (FRET), we showed that the initial 227 residues of LRMP and the N-terminus of HCN4 are necessary for LRMP to interact with HCN4. We found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. And we demonstrate that LRMP-regulation can be conferred to HCN2 by addition of the HCN4 N-terminus along with mutation of 5 residues in the S5 region and C-linker to the cognate HCN4 residues. Taken together, these results suggest that LRMP inhibits HCN4 through an isoform-specific interaction involving the N-terminals of both proteins that prevents the transduction of cAMP binding into a change in channel gating via an HCN4-specific orientation of the N-terminus, C-linker, and S4-S5 linker.

biophysics↗

bioRxiv2023

Dynamic landscape of the intracellular termini of ASIC1a

Acid-sensing ion channels (ASICs) are trimeric proton-gated sodium channels. Recently it has been shown that these channels play a role in necroptosis following prolonged acidic exposure like occurs in stroke. The C-terminus of the channel is thought to mediate necroptotic cell death through interaction with receptor interacting serine threonine kinase 1 (RIPK1). This interaction is hypothesized to be inhibited at rest via an interaction between the C-terminus and the N-terminus which blocks the RIPK1 binding site. Here, we use a combination of two transition metal ion FRET methods to investigate the conformational dynamics of the termini while the channel is closed and desensitized. We do not find evidence that the termini are close enough to be bound while the channel is at rest and find that the termini may modestly move closer together when desensitized. At rest, the N-terminus adopts a conformation parallel to the membrane about 10 [A] away. The distal end of the C-terminus may also spend time close to the membrane at rest. After acidification, the proximal portion of the N-terminus moves marginally closer to the membrane whereas the distal portion of the C-terminus swings away from the membrane. Together these data suggest that a new hypothesis for RIPK1 binding during stroke is needed.

biophysics↗

bioRxiv2021

Polyunsaturated fatty acid structure determines the strength of potentiation of Acid-sensing ion channels

Acid-sensing ion channels (ASICs) are sensitized to activation by inflammatory mediators like the polyunsaturated fatty acid (PUFA) arachidonic acid (AA). Previous work has shown that AA can potentiate ASIC currents at subsaturating proton concentrations, but the structural mechanisms of this change in gating are not understood. Here we show that PUFAs cause multiple gating changes in ASIC3 including shifting the pH dependence of activation, slowing the rate of desensitization, and increasing the current even at a saturating pH. The impact on gating depends on the nature of both the head and tail of the lipid with the head group structure primarily determining the magnitude of the effect on the channel. An N-acyl amino acid (NAAA), arachidonoyl glycine (AG), is such a strong regulator that it can act as a ligand at neutral pH. Mutation of an arginine in the outer segment of TM1 (R64) eliminated the effect of docosahexaenoic acid (DHA) even at high concentrations suggesting a potential interaction site for the lipid on the channel. Our results suggest a model where PUFAs bind to ASICs both via their tail group as well as an electrostatic interaction between the negatively charged PUFA head group and the positively charged arginine side chain. These data provide the first look at the structural features of lipids that are important for modulating ASICs and suggest a potential binding site for PUFAs on the channel. Short SummaryArachidonic acid (AA) is a known modulator of ASICs. Bankston and Klipp examine the structural requirements for ASIC modulation by a number of lipids related to AA. Negatively charged head groups are stronger potentiators and may interact with an arginine in TM1 near the outer leaflet of the plasma membrane.

biophysics↗

bioRxiv2020

Isoform-specific regulation of HCN4 channels by a family of endoplasmic reticulum proteins

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum transmembrane proteins that interact with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage-dependence of HCN4 activation in response to binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage-dependence of activation in the absence of cAMP. The mechanisms of action of LRMP and IRAG are novel; they are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 availability. Our results suggest important roles for LRMP and IRAG in regulation of cellular excitability and as tools for advancing mechanistic understanding of HCN4 channel function. Significance statementThe pore-forming subunits of ion channels are regulated by auxiliary interacting proteins. Hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channels are critical determinants of electrical excitability in many types of cells including neurons and cardiac pacemaker cells. Here we report the discovery of two novel HCN4 regulatory proteins. Despite their homology, the two proteins -- lymphoid-restricted membrane protein (LRMP) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG) -- have opposing effects on HCN4, causing loss- and gain-of-function, respectively. LRMP and IRAG are expected to play critical roles in regulation of physiological processes ranging from wakefulness to heart rate through their modulation of HCN4 channel function.

physiology↗

bioRxiv2019

Insights into the molecular mechanisms underlying the inhibition of acid-sensing ion channel 3 gating by Stomatin

Stomatin is a monotopic integral membrane protein found in all classes of life that has been shown to regulate members of the Acid-Sensing Ion Channel (ASIC) family. However, the mechanism by which Stomatin alters ASIC function is not known. Using chimeric channels, we combined patch clamp electrophysiology and FRET to search for regions of ASIC3 critical for binding to and regulation by Stomatin. With this approach, we found that regulation requires two distinct sites on ASIC3: the distal C-terminus and the first transmembrane domain. Mutation of the C-terminal site disrupts binding and regulation whereas disruption of the transmembrane site eliminates functional regulation. We then showed that Stomatin does not alter surface expression using fluorescence imaging and a surface biotinylation assay. Based on these findings, we propose a model whereby STOM is anchored to the channel via a site on the distal C-terminus but alters ASIC3 gating through action on TM1.

biophysics↗