Biology subjects

Chanda, B.

Publications and source records attributed to Chanda, B..

8 recordsLinked to original sources

bioRxiv2024

Lipid bilayers determine the allostery but not intrinsic affinity of cAMP binding to pacemaker channels

Cyclic adenosine monophosphate (cAMP), a second messenger, binds to hyperpolarization and cyclic nucleotide-gated (HCN) ion channels and regulates the automaticity of pacemaking activity. While cellular studies suggest that cAMP binding to HCN channels exhibits unusual cooperativity, recent findings using purified detergent-solubilized channels indicate independent binding to each subunit. This discrepancy raises the question of whether the lipid environment or endogenous cellular cofactors influence cAMP-dependent gating. To address this, we reconstituted purified human HCN channels in nanodiscs and resolved cAMP binding energetics at single-molecule resolution using nanophotonic waveguides. Our measurements reveal that, in contrast to detergent-solubilized channels, cAMP binds cooperatively to HCN channels reconstituted in a variety of lipid nanodiscs. Remarkably, the presence of lipid bilayer promotes ligand-binding allostery but not intrinsic binding affinity. To explore the molecular basis of bilayer-induced allostery, we determine the cryo-EM structure of HCN1 in soy polar lipid nanodiscs at a nominal resolution of 3.77 [A] resolution. Although the overall architecture is conserved, the average interfacial distance between the transmembrane domain and C-terminal domain of neighboring subunits are shorter in lipid nanodiscs. These findings indicate that the lipid bilayer regulates the function of pacemaker ion channels by enhancing inter-subunit interactions and underscore the fundamental role of membranes in amplifying the gating sensitivity of ion channels by promoting long-range cooperative interactions. Significance StatementLipid membranes are essential for the structure and function of membrane proteins, including ion channels. Lipid mimetics, such as non-ionic detergents, are widely used as surrogates for the membrane environment in structural and biophysical studies. Here, we demonstrate that while the overall structure of the pacemaker ion channel remains similar, lipid membranes--unlike detergents--promote cooperative ligand-binding transitions by modifying interactions at intersubunit interfaces. These findings provide new insights into the mechanism of ion channel regulation by lipid membranes.

biophysics↗

bioRxiv2024

DeFrND: detergent-free reconstitution into native nanodiscs with designer membrane scaffold peptides

Membrane scaffold proteins-based nanodiscs (NDs) have facilitated unprecedented structural and biophysical analysis of membrane proteins in a near-native lipid environment. However, successful reconstitution of membrane proteins in NDs requires prior solubilization and purification in detergents, which may impact their physiological structure and function. Furthermore, the detergent-mediated reconstitution of NDs is unlikely to recapitulate the precise composition or asymmetry of native membranes. To circumvent this fundamental limitation of traditional ND technology, we herein describe the development of membrane-solubilizing peptides to directly extract membrane proteins from native cell membranes into nanoscale discoids. By systematically protein engineering and screening, we created a new class of chemically modified Apolipoprotein-A1 mimetic peptides to enable the formation of detergent-free NDs (DeFrNDs) with high efficiency. NDs generated with these engineered membrane scaffold peptides are suitable for obtaining high-resolution structures using single-particle cryo-EM with native lipids. To further highlight the versatility of DeFrNDs, we directly extract a sampling of membrane signaling proteins with their surrounding native membranes for biochemical and biophysical interrogations.

biophysics↗

bioRxiv2024

Caveolar Compartmentalization is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure

BackgroundHeart rhythm relies on complex interactions between electrogenic membrane proteins and intracellular Ca2+ signaling in sinoatrial node (SAN) myocytes; however, mechanisms underlying the functional organization of proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, but the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND). MethodsBiochemical co-purification, in vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca2+ imaging, and immunofluorescent and electron microscopy analyses were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking. ResultsIn both mouse and human SANs, caveolae compartmentalized HCN4, Cav1.2, Cav1.3, Cav3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local SR Ca2+ release events (LCRs), which diffuse across [~]15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca2+ current, a negative shift of the HCN current (If) activation curve, and a 40% reduction of Na+/Ca2+-exchanger function, along with [~]2.3-times widening of the sarcolemma-SR distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extra-caveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by LCRs and trigger action potentials. HF remodeling mirrored caveolae disruption leading to NCX1-LCR uncoupling and SND. ConclusionsSAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, potentially demonstrating a new dimension of SAN remodeling and providing a newly recognized target for therapy.

physiology↗

bioRxiv2023

Structural Basis for Hyperpolarization-dependent Opening of the Human HCN1 Channel

Hyperpolarization and cyclic-nucleotide (HCN) activated ion channels play a critical role in generating self-propagating action potentials in pacemaking and rhythmic electrical circuits in the human body. Unlike most voltage-gated ion channels, the HCN channels activate upon membrane hyperpolarization, but the structural mechanisms underlying this gating behavior remain unclear. Here, we present cryo-electron microscopy structures of human HCN1 in Closed, Intermediate, and Open states. Our structures reveal that the inward motion of two gating charges past the charge transfer center (CTC) and concomitant tilting of the S5 helix drives the opening of the central pore. In the intermediate state structure, a single gating charge is positioned below the CTC and the pore appears closed, whereas in the open state structure, both charges move past CTC and the pore is fully open. Remarkably, the downward motion of the voltage sensor is accompanied by progressive unwinding of the inner end of S4 and S5 helices disrupting the tight gating interface that stabilizes the Closed state structure. This "melting" transition at the intracellular gating interface leads to a concerted iris-like displacement of S5 and S6 helices, resulting in pore opening. These findings reveal key structural features that are likely to underlie reversed voltage-dependence of HCN channels.

biochemistry↗

bioRxiv2022

Charge-voltage curves of Shaker potassium channel are not hysteretic at steady state

Charged residues in transmembrane segments of voltage-gated ion channels (VGICs) sense and respond to changes in the electric field. The movement of these gating charges underpins voltage-dependent activation and is also a direct metric of the net free-energy of channel activation. However, for most voltage-gated ion channels, the charge-voltage (Q-V) curves appear to be dependent on initial conditions. For instance, Q-V curves of Shaker potassium channel obtained by hyperpolarizing from 0 mV is left shifted compared to those obtained by depolarizing from a holding potential of -80 mV. This hysteresis in Q-V curves is a common feature of channels in the VGIC superfamily and raises profound questions about channel energetics because the net free-energy of channel gating is a state function and should be path independent. Due to technical limitations, conventional gating current protocols are limited to test pulse durations of <500 ms which raises the possibility that the dependence of Q-V on initial conditions reflects a lack of equilibration. Others have suggested that the hysteresis is fundamental thermodynamic property of voltage-gated ion channels and reflects energy dissipation due to measurements under non-equilibrium conditions inherent to rapid voltage jumps (Villalba-Galea, 2017). Using an improved gating current and voltage-clamp fluorometry protocols, we show that the gating hysteresis arising from different initial conditions in Shaker potassium channel is eliminated with ultra-long (18-25 second) test pulses. Our study identifies a modified gating current recording protocol to obtain steady state Q-V curves of a voltage-gated ion channel. Above all, these findings demonstrate that the gating hysteresis in Shaker channel is a kinetic phenomenon rather than a true thermodynamic property of the channel and the charge-voltage curve is a true measure of the net-free energy of channel gating.

biophysics↗

bioRxiv2022

Interplay between VSD, pore and membrane lipids in electromechanical coupling in HCN channels

Hyperpolarized-activated and Cyclic Nucleotide-gated (HCN) channels are the only members of the voltage-gated ion channel superfamily in mammals that open upon hyperpolarization, conferring them pacemaker properties that are instrumental for rhythmic firing of cardiac and neuronal cells. Activation of their voltage-sensor domains (VSD) upon hyperpolarization occurs through a downward movement of the S4 helix bearing the gating charges, which triggers a break in the alpha-helical hydrogen bonding pattern at the level of a conserved Serine residue. Previous structural and molecular simulation studies had however failed to capture pore opening that should be triggered by VSD activation, presumably because of a low VSD/pore electromechanical coupling efficiency and the limited timescales accessible to such techniques. Here, we have used advanced modeling strategies, including enhanced sampling molecular dynamics simulations exploiting comparisons between non-domain swapped voltage-gated ion channel structures trapped in closed and open states to trigger pore gating and characterize electromechanical coupling in HCN1. We propose that the coupling mechanism involves the reorganization of the interfaces between the VSD helices, in particular S4, and the pore-forming helices S5 and S6, subtly shifting the balance between hydrophobic and hydrophilic interactions in a "domino effect" during activation and gating in this region. Remarkably, our simulations reveal state-dependent occupancy of lipid molecules at this emergent coupling interface, suggesting a key role of lipids in hyperpolarization-dependent gating. Our model provides a rationale for previous observations and a possible mechanism for regulation of HCN channels by the lipidic components of the membrane.

biophysics↗

bioRxiv2019

High-Throughput Single-Molecule Analysis via Divisive Segmentation and Clustering

Single-molecule approaches provide insight into the dynamics of biomolecules, yet analysis methods have not scaled with the growing size of data sets acquired in high-throughput experiments. We present a new analysis platform (DISC) that uses divisive clustering to accelerate unsupervised analysis of single-molecule trajectories by up to three orders of magnitude with improved accuracy. Using DISC, we reveal an inherent lack of cooperativity between cyclic nucleotide binding domains from HCN pacemaker ion channels embedded in nanophotonic zero-mode waveguides.

biophysics↗

bioRxiv2018

Bipolar Switching by HCN Voltage Sensor Underlies Hyperpolarization Activation.

Despite sharing a common architecture with archetypal voltage-gated ion channels (VGIC), the hyperpolarization- and cyclic AMP-activated ion (HCN) channels open upon hyperpolarization rather than depolarization. The basic motions of voltage sensor and pore gates are conserved implying that these domains are inversely coupled in HCN channels. Using structure-guided protein engineering, we systematically assembled an array of mosaic channels that display the full complement of voltage-activation phenotypes observed in the VGIC superfamily. Our studies reveal that the voltage-sensing S3b-S4 transmembrane segment of the HCN channel has an intrinsic ability to drive pore opening in either direction. Specific contacts at the pore-voltage sensor interface and unique interactions near the pore gate forces the HCN channel into a hERG-like inactivated state, thereby obscuring their opening upon depolarization. Our findings reveal an unexpected common principle underpinning voltage gating in the VGIC superfamily and identify the essential determinants of gating polarity.

neuroscience↗