Structure and reconstitution of a MCU-EMRE mitochondrial Ca2+ uniporter complex

The proteins MCU and EMRE form the minimal functional unit of the mitochondrial calcium uniporter complex in metazoans, a highly selective and tightly controlled Ca2+ channel of the inner mitochondrial membrane that regulates cellular metabolism. Here we present functional reconstitution of an MCU-EMRE complex from the red flour beetle, Tribolium castaneum, and a cryo-EM structure of the complex at 3.5 Å resolution. Robust Ca2+ uptake is observed into proteoliposomes containing the purified complex and is dependent on EMRE. The structure reveals a tetrameric channel with a single ion pore. EMRE is located at the periphery of the transmembrane domain and associates primarily with the first transmembrane helix of MCU. Coiled coil and juxtamembrane domains within the matrix portion of the complex adopt markedly different conformations than in a structure of a human MCU-EMRE complex, suggesting that the structures represent different conformations of these functionally similar metazoan channels.

Ion transport across solid-state ion channels perturbed by directed strain

Using computer simulations, we demonstrate ion permeation measurements across strained membranes that may potentially be used to obtain directional profiles of ion-pore energetics as contributed by the pore edge atoms.

Cadmium opens GluK2 kainate receptors with cysteine substitutions at the M3 helix bundle crossing

Kainate receptors are ligand-gated ion channels that have two major roles in the central nervous system: they mediate a postsynaptic component of excitatory neurotransmission at some glutamatergic synapses and modulate transmitter release at both excitatory and inhibitory synapses. Accumulating evidence implicates kainate receptors in a variety of neuropathologies, including epilepsy, psychiatric disorders, developmental delay, and cognitive impairment. Here, to gain a deeper understanding of the conformational changes associated with agonist binding and channel opening, we generate a series of Cys substitutions in the GluK2 kainate receptor subunit, focusing on the M3 helices that line the ion pore and form the bundle-crossing gate at the extracellular mouth of the channel. Exposure to 50 µM Cd produces direct activation of homomeric mutant channels bearing Cys substitutions in (A657C), or adjacent to (L659C), the conserved SYTANLAAF motif. Activation by Cd is occluded by modification with 2-aminoethyl MTS (MTSEA), indicating that Cd binds directly and specifically to the substituted cysteines. Cd potency for the A657C mutation (EC50 = 10 µM) suggests that binding involves at least two coordinating residues, whereas weaker Cd potency for L659C (EC50 = 2 mM) implies that activation does not require tight coordination by multiple side chains for this substitution. Experiments with heteromeric and chimeric channels indicate that activation by Cd requires Cys substitution at only two of the four subunits within a tetrameric receptor and that activation is similar for substitution within subunits in either the A/C or B/D conformations. We develop simple kinetic models for the A657C substitution that reproduce several features of Cd activation as well as the low-affinity inhibition observed at higher Cd concentrations (5–20 mM). Together, these results demonstrate rapid and reversible channel activation, independent of agonist site occupancy, upon Cd binding to Cys side chains at two specific locations along the GluK2 inner helix.

An allosteric mechanism of inactivation in the calcium-dependent chloride channel BEST1

Bestrophin proteins are calcium (Ca2+)-activated chloride channels. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Whole-cell recordings show that ionic currents through BEST1 run down over time, but it is unclear whether this behavior is intrinsic to the channel or the result of cellular factors. Here, using planar lipid bilayer recordings of purified BEST1, we show that current rundown is an inherent property of the channel that can now be characterized as inactivation. Inactivation depends on the cytosolic concentration of Ca2+, such that higher concentrations stimulate inactivation. We identify a C-terminal inactivation peptide that is necessary for inactivation and dynamically interacts with a receptor site on the channel. Alterations of the peptide or its receptor dramatically reduce inactivation. Unlike inactivation peptides of voltage-gated channels that bind within the ion pore, the receptor for the inactivation peptide is on the cytosolic surface of the channel and separated from the pore. Biochemical, structural, and electrophysiological analyses indicate that binding of the peptide to its receptor promotes inactivation, whereas dissociation prevents it. Using additional mutational studies we find that the “neck” constriction of the pore, which we have previously shown to act as the Ca2+-dependent activation gate, also functions as the inactivation gate. Our results indicate that unlike a ball-and-chain inactivation mechanism involving physical occlusion of the pore, inactivation in BEST1 occurs through an allosteric mechanism wherein binding of a peptide to a surface-exposed receptor controls a structurally distant gate.

TRPC5 Does Not Cause or Aggravate Glomerular Disease

Transient receptor potential channel 5 (TRPC5) is highly expressed in brain and kidney and mediates calcium influx and promotes cell migration. In the kidney, loss of TRPC5 function has been reported to benefit kidney filter dynamics by balancing podocyte cytoskeletal remodeling. However, in vivo gain-in-function studies of TRPC5 with respect to kidney function have not been reported. To address this gap, we developed two transgenic mouse models on the C57BL/6 background by overexpressing either wild-type TRPC5 or a TRPC5 ion-pore mutant. Compared with nontransgenic controls, neither transgenic model exhibited an increase in proteinuria at 8 months of age or a difference in LPS-induced albuminuria. Moreover, activation of TRPC5 by Englerin A did not stimulate proteinuria, and inhibition of TRPC5 by ML204 did not significantly lower the level of LPS-induced proteinuria in any group. Collectively, these data suggest that the overexpression or activation of the TRPC5 ion channel does not cause kidney barrier injury or aggravate such injury under pathologic conditions.

On the permeation of large organic cations through the pore of ATP-gated P2X receptors

Pore dilation is thought to be a hallmark of purinergic P2X receptors. The most commonly held view of this unusual process posits that under prolonged ATP exposure the ion pore expands in a striking manner from an initial small-cation conductive state to a dilated state, which allows the passage of larger synthetic cations, such as N-methyl-d-glucamine (NMDG+). However, this mechanism is controversial, and the identity of the natural large permeating cations remains elusive. Here, we provide evidence that, contrary to the time-dependent pore dilation model, ATP binding opens an NMDG+-permeable channel within milliseconds, with a conductance that remains stable over time. We show that the time course of NMDG+ permeability superimposes that of Na+ and demonstrate that the molecular motions leading to the permeation of NMDG+ are very similar to those that drive Na+ flow. We found, however, that NMDG+ “percolates” 10 times slower than Na+ in the open state, likely due to a conformational and orientational selection of permeating molecules. We further uncover that several P2X receptors, including those able to desensitize, are permeable not only to NMDG+ but also to spermidine, a large natural cation involved in ion channel modulation, revealing a previously unrecognized P2X-mediated signaling. Altogether, our data do not support a time-dependent dilation of the pore on its own but rather reveal that the open pore of P2X receptors is wide enough to allow the permeation of large organic cations, including natural ones. This permeation mechanism has considerable physiological significance.

Binding site opening by loop C shift and chloride ion-pore interaction in the GABAAreceptor model

Molecular dynamics simulations of the shut α1β2γ2GABAAheteropentamer receptor homology model reveal significant differences between intersubunit interfaces (ligand binding G1, G2 and non-binding) compared to homomeric receptor assemblies and possible ion interaction sites in the top part of the transmembrane domain (TMD).