130 research outputs found
Cholesterol Sensitivity of Kir2.1 is Modulated by a Cytosolic Belt: Implications for Kir Gating
The Molecular Mechanism by which PIP2 Opens the Intracellular G-Loop Gate of a Kir3.1 Channel
Abstract Inwardly rectifying potassium (Kir) channels are characterized by a long pore comprised of continuous transmembrane and cytosolic portions. A high-resolution structure of a Kir3.1 chimera revealed the presence of the cytosolic (G-loop) gate captured in the closed or open conformations. Here, we conducted molecular-dynamics simulations of these two channel states in the presence and absence of phosphatidylinositol bisphosphate (PIP2), a phospholipid that is known to gate Kir channels. Simulations of the closed state with PIP2 revealed an intermediate state between the closed and open conformations involving direct transient interactions with PIP2, as well as a network of transitional inter- and intrasubunit interactions. Key elements in the G-loop gating transition involved a PIP2-driven movement of the N-terminus and C-linker that removed constraining intermolecular interactions and led to CD-loop stabilization of the G-loop gate in the open state. To our knowledge, this is the first dynamic molecular view of PIP2-induced channel gating that is consistent with existing experimental data
Identification of a Novel PIP2 Interaction Site and its Allosteric Regulation by the RCK1 Site Associated with Ca2+ Coordination in Slo1 Channels
We consider the Schr\"odinger operator on a combinatorial graph consisting of
a finite graph and a finite number of discrete half-lines, all jointed
together, and compute an asymptotic expansion of its resolvent around the
threshold . Precise expressions are obtained for the first few coefficients
of the expansion in terms of the generalized eigenfunctions. This result
justifies the classification of threshold types solely by growth properties of
the generalized eigenfunctions. By choosing an appropriate free operator a
priori possessing no zero eigenvalue or zero resonance we can simplify the
expansion procedure as much as that on the single discrete half-line.Comment: 55 pages, minor revisions, final versio
Elucidation of molecular kinetic schemes from macroscopic traces using system identification
Overall cellular responses to biologically-relevant stimuli are mediated by networks of simpler lower-level processes. Although information about some of these processes can now be obtained by visualizing and recording events at the molecular level, this is still possible only in especially favorable cases. Therefore the development of methods to extract the dynamics and relationships between the different lower-level (microscopic) processes from the overall (macroscopic) response remains a crucial challenge in the understanding of many aspects of physiology. Here we have devised a hybrid computational-analytical method to accomplish this task, the SYStems-based MOLecular kinetic scheme Extractor (SYSMOLE). SYSMOLE utilizes system-identification input-output analysis to obtain a transfer function between the stimulus and the overall cellular response in the Laplace-transformed domain. It then derives a Markov-chain state molecular kinetic scheme uniquely associated with the transfer function by means of a classification procedure and an analytical step that imposes general biological constraints. We first tested SYSMOLE with synthetic data and evaluated its performance in terms of its rate of convergence to the correct molecular kinetic scheme and its robustness to noise. We then examined its performance on real experimental traces by analyzing macroscopic calcium-current traces elicited by membrane depolarization. SYSMOLE derived the correct, previously known molecular kinetic scheme describing the activation and inactivation of the underlying calcium channels and correctly identified the accepted mechanism of action of nifedipine, a calcium-channel blocker clinically used in patients with cardiovascular disease. Finally, we applied SYSMOLE to study the pharmacology of a new class of glutamate antipsychotic drugs and their crosstalk mechanism through a heteromeric complex of G protein-coupled receptors. Our results indicate that our methodology can be successfully applied to accurately derive molecular kinetic schemes from experimental macroscopic traces, and we anticipate that it may be useful in the study of a wide variety of biological systems
A Computational Model Reveals the Action of Gβγ at an Inter-Subunit Cleft to Activate GIRK1 Channels
The atrial G protein-regulated inwardly rectifying K+ (GIRK1 and GIRK4) heterotetrameric channels underlie the acetylcholine-induced K+ current responsible for vagal inhibition of heart rate and are activated by the G protein βγ subunits (Gβγ). We used a multistage protein-protein docking approach with data from published structures of GIRK1 and Gβγ to generate an experimentally testable interaction model of Gβγ docked onto the cytosolic domains of the GIRK1 homotetramer. The model suggested a mechanism by which Gβγ promotes the open state of a specific cytosolic gate in the channel, the G-loop gate. The predicted structure showed that the Gβ subunit interacts with the channel near the site of action for ethanol and stabilizes an intersubunit cleft formed by two loops (LM and DE) of adjacent channel subunits. Using a heterologous expression system, we disrupted the predicted GIRK1- and Gβγ-interacting residues by mutation of one protein and then rescued the regulatory activity by mutating reciprocal residues in the other protein. Disulfide crosslinking of channels and Gβγ subunits with cysteine mutations at the predicted interacting residues yielded activated channels. The mechanism of Gβγ-induced activation of GIRK4 was distinct from GIRK1 homotetramers. However, GIRK1-GIRK4 heteroterameric channels activated by Gβγ displayed responses indicating that the GIRK1 subunit dominated the response pattern. This work demonstrated that combining computational with experimental approaches is an effective method for elucidating interactions within protein complexes that otherwise might be challenging to decipher
An analytical tool for elucidating ion-channel molecular mechanisms from macroscopic current traces
Building models to describe the dynamics of macroscopic currents through ion channels has been the object of numerous studies in the literature with the aim of understanding ion-channel function. Following a perturbation, typically a step in voltage or ligand concentration, the response is formed by a combination of different processes such as activation or inactivation that pull the measured quantity (macroscopic current) in the same or opposite directions with different strengths and different time constants. Although this dynamic response can be readily recorded in time, the relationship between the underlying processes cannot be easily teased apart without structural analysis or single-channel recordings. An example is the classic problem of determining from sodium-channel macroscopic traces whether the activation and inactivation processes occur in parallel or inactivation is dependent on previous activation.
We present a mathematical tool to analyze electrophysiological traces and derive molecular kinetic schemes that reflect the interplay between the different processes involved. This tool is based on system-identification algorithms and consists of three modules as summarized in Figure 1. The identifier takes the input and output signals in the time domain and applies autoregressive ARX methods to obtain a transfer function in the Laplace domain yielding a set of poles, zeros and gain that provide a unique signature of the channel response. The classifier capitalizes on this signature to reveal the block diagram associated with the interplay of the processes, that are here described as first order systems in classic engineering terms (a relaxation with one time constant and a gain for each process). Finally, the molecular kinetic converter uses the transfer function together with the block diagram and maps them into a molecular kinetic scheme, a description with states associated with a system of differential equations
G protein-coupled receptor-effector macromolecular membrane assemblies (GEMMAs)
G protein-coupled receptors (GPCRs) are the largest group of receptors involved in cellular signaling across the plasma membrane and a major class of drug targets. The canonical model for GPCR signaling involves three components the GPCR, a heterotrimeric G protein and a proximal plasma membrane effector that have been generally thought to be freely mobile molecules able to interact by 'collision coupling'. Here, we synthesize evidence that supports the existence of GPCR-effector macromolecular membrane assemblies (GEMMAs) comprised of specific GPCRs, G proteins, plasma membrane effector molecules and other associated transmembrane proteins that are pre-assembled prior to receptor activation by agonists, which then leads to subsequent rearrangement of the GEMMA components. The GEMMA concept offers an alternative and complementary model to the canonical collision-coupling model, allowing more efficient interactions between specific signaling components, as well as the integration of the concept of GPCR oligomerization as well as GPCR interactions with orphan receptors, truncated GPCRs and other membrane-localized GPCR-associated proteins. Collision-coupling and pre-assembled mechanisms are not exclusive and likely both operate in the cell, providing a spectrum of signaling modalities which explains the differential properties of a multitude of GPCRs in their different cellular environments. Here, we explore the unique pharmacological characteristics of individual GEMMAs, which could provide new opportunities to therapeutically modulate GPCR signaling
Molecular overlap in the regulation of SK channels by small molecules and phosphoinositides
Phosphatidylinositol 4,5-bisphosphate (PIP2) directly interacts with the small-conductance Ca2+-activated K+2-a (SK2-a) channel/calmodulin complex, serving as a critical element in the regulation of channel activity. We report that changes of protein conformation in close proximity to the PIP2 binding site induced by a small-molecule SK channel modulator, NS309, can effectively enhance the interaction between the protein and PIP2 to potentiate channel activity. This novel modulation of PIP2 sensitivity by small-molecule drugs is likely not to be limited in its application to SK channels, representing an intriguing strategy to develop drugs controlling the activity of the large number of PIP2-dependent proteins
Requirement for an Activated G Protein α (Gα) Subunit for Gβγ Activation of a Purified Mammalian GIRk1 Channel Reconstituted in Planar Lipid Bilayers
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