Application of a Statistical and Linear Response Theory to Multi-Ion Na+ Conduction in NaChBac.

Biological ion channels are fundamental to maintaining life. In this manuscript we apply our recently developed statistical and linear response theory to investigate Na+ conduction through the prokaryotic Na+ channel NaChBac. This work is extended theoretically by the derivation of ionic conductivity and current in an electrochemical gradient, thus enabling us to compare to a range of whole-cell data sets performed on this channel. Furthermore, we also compare the magnitudes of the currents and populations at each binding site to previously published single-channel recordings and molecular dynamics simulations respectively. In doing so, we find excellent agreement between theory and data, with predicted energy barriers at each of the four binding sites of ∼4,2.9,3.6, and 4kT.

Changes in Ion Selectivity Following the Asymmetrical Addition of Charge to the Selectivity Filter of Bacterial Sodium Channels.

Voltage-gated sodium channels (NaVs) play fundamental roles in eukaryotes, but their exceptional size hinders their structural resolution. Bacterial NaVs are simplified homologues of their eukaryotic counterparts, but their use as models of eukaryotic Na+ channels is limited by their homotetrameric structure at odds with the asymmetric Selectivity Filter (SF) of eukaryotic NaVs. This work aims at mimicking the SF of eukaryotic NaVs by engineering radial asymmetry into the SF of bacterial channels. This goal was pursued with two approaches: the co-expression of different monomers of the NaChBac bacterial channel to induce the random assembly of heterotetramers, and the concatenation of four bacterial monomers to form a concatemer that can be targeted by site-specific mutagenesis. Patch-clamp measurements and Molecular Dynamics simulations showed that an additional gating charge in the SF leads to a significant increase in Na+ and a modest increase in the Ca2+ conductance in the NavMs concatemer in agreement with the behavior of the population of random heterotetramers with the highest proportion of channels with charge -5e. We thus showed that charge, despite being important, is not the only determinant of conduction and selectivity, and we created new tools extending the use of bacterial channels as models of eukaryotic counterparts.

CaV1.2 and CaV1.3 voltage-gated L-type Ca2+ channels in rat white fat adipocytes.

L-type channel antagonists are of therapeutic benefit in the treatment of hyperlipidaemia and insulin resistance. Our aim was to identify L-type voltage-gated Ca2+ channels in white fat adipocytes, and determine if they affect intracellular Ca2+, lipolysis and lipogenesis. We used a multidisciplinary approach of molecular biology, confocal microscopy, Ca2+ imaging and metabolic assays to explore this problem using adipocytes isolated from adult rat epididymal fat pads. CaV1.2, CaV1.3 and CaV1.1 alpha1, beta and alpha2delta subunits were detected at the gene expression level. The CaV1.2 and CaV1.3 alpha1 subunits were identified in the plasma membrane at the protein level. Confocal microscopy with fluorescent antibodies labelled CaV1.2 in the plasma membrane. Ca2+ imaging revealed that the intracellular Ca2+ concentration, [Ca2 +]i was reversibly decreased by removal of extracellular Ca2+, an effect mimicked by verapamil, nifedipine and Co2+, all blockers of L-type channels, whereas the Ca2+ channel agonist BAY-K8644 increased [Ca2+]i. The finding that the magnitude of these effects correlated with basal [Ca2+]i suggests that adipocyte [Ca2+]i is controlled by L-type Ca2+ channels that are constitutively active at the adipocyte depolarized membrane potential. Pharmacological manipulation of L-type channel activity modulated both basal and catecholamine-stimulated lipolysis but not insulin-induced glucose uptake or lipogenesis. We conclude that white adipocytes have constitutively active L-type Ca2+ channels which explains their sensitivity of lipolysis to Ca2+ channel modulators. Our data suggest CaV1.2 as a potential novel therapeutic target in the treatment of obesity.

Covalent linkage of bacterial voltage-gated sodium channels.

BACKGROUND: Bacterial sodium channels are important models for understanding ion permeation and selectivity. However, their homotetrameric structure limits their use as models for understanding the more complex eukaryotic voltage-gated sodium channels (which have a pseudo-heterotetrameric structure formed from an oligomer composed of four domains). To bridge this gap we attempted to synthesise oligomers made from four covalently linked bacterial sodium channel monomers and thus resembling their eukaryotic counterparts. RESULTS: Western blot analyses revealed NaChBac oligomers to be inherently unstable whereas intact expression of NavMs oligomers was possible. Immunodectection using confocal microscopy and electrophysiological characterisation of NavMs tetramers confirmed plasma membrane localisation and equivalent functionality with wild type NavMs channels when expressed in human embryonic kidney cells. CONCLUSION: This study has generated new tools for the investigation of eukaryotic channels. The successful covalent linkage of four bacterial Nav channel monomers should permit the introduction of radial asymmetry into the structure of bacterial Nav channels and enable the known structures of these channels to be used to gain unique insights into structure-function relationships of their eukaryotic counterparts.

Different roles for aspartates and glutamates for cation permeation in bacterial sodium channels.

A key driving force for ion channel selectivity is represented by the negative charge of the Selectivity Filter carried by aspartate (D) and glutamate (E) residues. However, the structural effects and specific properties of D and E residues have not been extensively studied. In order to investigate this issue we studied the mutants of NaChBac channel with all possible combinations of D and E in the charged rings in position 191 and 192. Electrophysiological measurements showed significant Ca2+ currents only when position 191 was occupied by E. Equilibrium Molecular Dynamics simulations revealed the existence of two binding sites, corresponding to the charged rings and another one, more internal, at the level of L190. The simulations showed that the ion in the innermost site can interact with the residue in position 191 only when this is glutamate. Based on the MD simulations, we suggest that a D in position 191 leads to a high affinity Ca2+ block site resulting from a significant drop in the free energy of binding for an ion moving between the binding sites; in contrast, the free energy change is more gradual when an E residue occupies position 191, resulting in Ca2+ permeability. This scenario is consistent with the model of ion channel selectivity through stepwise changes in binding affinity proposed by Dang and McCleskey. Our study also highlights the importance of the structure of the selectivity filter which should contribute to the development of more detailed physical models for ion channel selectivity.

On the selectivity of the NaChBac channel: an integrated computational and experimental analysis of sodium and calcium permeation.

Ion channel selectivity is essential for their function, yet the molecular basis of a channel's ability to select between ions is still rather controversial. In this work, using a combination of molecular dynamics simulations and electrophysiological current measurements we analyze the ability of the NaChBac channel to discriminate between calcium and sodium. Our simulations show that a single calcium ion can access the Selectivity Filter (SF) interacting so strongly with the glutamate ring so as to remain blocked inside. This is consistent with the tiny calcium currents recorded in our patch-clamp experiments. Two reasons explain this scenario. The first is the higher free energy of ion/SF binding of Ca2+ with respect to Na+. The second is the strong electrostatic repulsion exerted by the resident ion that turns back a second potentially incoming Ca2+, preventing the knock-on permeation mechanism. Finally, we analyzed the possibility of the Anomalous Mole Fraction Effect (AMFE), i.e. the ability of micromolar Ca2+ concentrations to block Na+ currents. Current measurements in Na+/Ca2+ mixed solutions excluded the AMFE, in agreement with metadynamics simulations showing the ability of a sodium ion to by-pass and partially displace the resident calcium. Our work supports a new scenario for Na+/Ca2+ selectivity in the bacterial sodium channel, challenging the traditional notion of an exclusion mechanism strictly confining Ca2+ ions outside the channel.

Sodium Binding Sites and Permeation Mechanism in the NaChBac Channel: A Molecular Dynamics Study.

NaChBac was the first discovered bacterial sodium voltage-dependent channel, yet computational studies are still limited due to the lack of a crystal structure. In this work, a pore-only construct built using the NavMs template was investigated using unbiased molecular dynamics and metadynamics. The potential of mean force (PMF) from the unbiased run features four minima, three of which correspond to sites IN, CEN, and HFS discovered in NavAb. During the run, the selectivity filter (SF) is spontaneously occupied by two ions, and frequent access of a third one is often observed. In the innermost sites IN and CEN, Na+ is fully hydrated by six water molecules and occupies an on-axis position. In site HFS sodium interacts with a glutamate and a serine from the same subunit and is forced to adopt an off-axis placement. Metadynamics simulations biasing one and two ions show an energy barrier in the SF that prevents single-ion permeation. An analysis of the permeation mechanism was performed both computing minimum energy paths in the axial-axial PMF and through a combination of Markov state modeling and transition path theory. Both approaches reveal a knock-on mechanism involving at least two but possibly three ions. The currents predicted from the unbiased simulation using linear response theory are in excellent agreement with single-channel patch-clamp recordings.

Ion channels of the nuclear membrane of hippocampal neurons.

Rise in Ca(2+) concentration in the nucleus affects gene transcription and has been implicated in neuroprotection, transcription-dependent neuronal plasticity, and pain modulation, but the mechanism of regulation of nuclear Ca(2+) remains poorly understood. The nuclear envelope is a part of the endoplasmic reticulum and may be one of the sources of nuclear Ca(2+) . Here, we studied ion channels in the nuclear membrane of hippocampal neurons using the patch-clamp technique. We have found that the nuclear membrane of CA1 pyramidal and dentate gyrus granule (DG), but not CA3 pyramidal neurons, was enriched in functional inositol 1,4,5-trisphosphate receptors/Ca(2+) -release channels (IP3 Rs) localized mainly in the inner nuclear membrane. A single nuclear ryanodine receptor (RyR) has been detected only in DG granule neurons. Nuclei of the hippocampal neurons also expressed a variety of spontaneously active cation and anion channels specific for each type of neuron. In particular, large-conductance ion channels selective for monovalent cations (LCC) were coexpressed with IP3 Rs. These data suggest that: (1) the nuclear membranes of hippocampal neurons contain distinct sets of ion channels, which are specific for each type of neuron; (2) IP3 Rs, but not RyRs are targeted to the inner nuclear membrane of CA1 pyramidal and DG granule, but they were not found in the nuclear membranes of CA3 pyramidal neurons; (3) the nuclear envelope of these neurons is specialized to release Ca(2+) into the nucleoplasm which may amplify Ca(2+) signals entering the nucleus from the cytoplasm or generate Ca(2+) transients on its own; (4) LCC channels are an integral part the of Ca(2+) -releasing machinery providing a route for counterflow of К(+) and thereby facilitating Ca(2+) movement in and out of the Ca(2+) store.

Intracellular calcium channels: inositol-1,4,5-trisphosphate receptors.

The inositol-1,4,5-trisphosphate receptors (InsP3Rs) are the major intracellular Ca(2+)-release channels in cells. Activity of InsP3Rs is essential for elementary and global Ca(2+) events in the cell. There are three InsP3Rs isoforms that are present in mammalian cells. In this review we will focus primarily on InsP3R type 1. The InsP3R1 is a predominant isoform in neurons and it is the most extensively studied isoform. Combination of biophysical and structural methods revealed key mechanisms of InsP3R function and modulation. Cell biological and biochemical studies lead to identification of a large number of InsP3R-binding proteins. InsP3Rs are involved in the regulation of numerous physiological processes, including learning and memory, proliferation, differentiation, development and cell death. Malfunction of InsP3R1 play a role in a number of neurodegenerative disorders and other disease states. InsP3Rs represent a potentially valuable drug target for treatment of these disorders and for modulating activity of neurons and other cells. Future studies will provide better understanding of physiological functions of InsP3Rs in health and disease.