Unlocking the brain's zinc code: implications for cognitive function and disease.

Zn transport across neuronal membranes relies on two classes of transition metal transporters: the ZnT (SLC30) and ZIP (SLC39) families. These proteins function to decrease and increase cytosolic Zn levels, respectively. Dysfunction of ZnT and ZIP transporters can alter intracellular Zn levels resulting in deleterious effects.

Single-molecule quantification of the oligomeric state of ZIP transporters in mammalian cells with fluorescence correlation spectroscopy.

The SLC39 family of transporters, otherwise known as ZIPs for Zrt and Irt-like Proteins, function to increase cytosolic levels of transition metals. ZIP transporters have been identified at all phylogenetic levels and are members of the SoLute Carrier (SLC) superfamily. There are fourteen ZIP transporters encoded in the human genome.

Heterologous Expression of Full-Length and Truncated Human ZIP4 Zinc Transporter in .

The human (h) transporter hZIP4 is the primary Zn importer in the intestine. hZIP4 is also expressed in a variety of organs such as the pancreas and brain. Dysfunction of hZIP4 can result in the Zn deficiency disease acrodermatitis enteropathica (AE).

Channelrhodopsin C1C2: Photocycle kinetics and interactions near the central gate.

Channelrhodopsins (ChR) are light-sensitive cation channels used in optogenetics, a technique that applies light to control cells (e.g., neurons) that have been modified genetically to express those channels.

Characterizing Channelrhodopsin Channel Properties Via Two-Electrode Voltage Clamp and Kinetic Modeling.

Two-electrode voltage clamp (TEVC) is a preferred electrophysiological technique used to study gating kinetics and ion selectivity of light-activated channelrhodopsins (ChRs). The method uses two intracellular microelectrodes to hold, or clamp, the membrane potential at a specific value and measure the flow of ions across the plasma membrane. Here, we describe the use of TEVC and a simple solution exchange protocol to measure cation selectivity and analyze gating kinetics of the C1C2 chimera expressed in Xenopus laevis oocytes.

Quantifying the Oligomeric State of hZIP4 on the Surface of Cells.

The human (h) zinc transporter ZIP4 is expressed on the plasma membrane and functions to increase cytosolic zinc levels. Mutations in hZIP4 cause the disease acrodermatitis enteropathica. Dysfunction in the regulation of hZIP4 has also been indicated in solid tissue cancers, including pancreatic and prostate cancer.

Concomitant disorder and high-affinity zinc binding in the human zinc- and iron-regulated transport protein 4 intracellular loop.

The human zinc- and iron-regulated transport protein 4 (hZIP4) protein is the major plasma membrane protein responsible for the uptake of zinc in the body, and as such it plays a key role in cellular zinc homeostasis. hZIP4 plasma membrane levels are regulated through post-translational modification of its large, disordered, histidine-rich cytosolic loop (ICL2) in response to intracellular zinc concentrations. Here, structural characteristics of the isolated disordered loop region, both in the absence and presence of zinc, were investigated using nuclear magnetic resonance (NMR) spectroscopy.

An optimized and automated approach to quantifying channelrhodopsin photocurrent kinetics.

Channelrhodopsins are light-activated ion channels that enable targetable activation or inhibition of excitable cells with light. Ion conductance can generally be described by a four step photocycle, which includes two open and two closed states. While a complete understanding of channelrhodopsin function cannot be understood in the absence of kinetic modeling, model fitting requires manual fitting, which is laborious and technically complicated for non-experts.

The emerging role of zinc transporters in cellular homeostasis and cancer.

Zinc is an essential micronutrient that plays a role in the structural or enzymatic functions of many cellular proteins. Cellular zinc homeostasis involves the opposing action of two families of metal transporters: the ZnT (SLC30) family that functions to reduce cytoplasmic zinc concentrations and the ZIP (SLC39) family that functions to increase cytoplasmic zinc concentrations. Fluctuations in intracellular zinc levels mediated by these transporter families affect signaling pathways involved in normal cell development, growth, differentiation and death.

Adjacent channelrhodopsin-2 residues within transmembranes 2 and 7 regulate cation selectivity and distribution of the two open states.

Channelrhodopsin-2 (ChR2) is a light-activated channel that can conduct cations of multiple valencies down the electrochemical gradient. Under continuous light exposure, ChR2 transitions from a high-conducting open state (O1) to a low-conducting open state (O2) with differing ion selectivity. The molecular basis for the O1 → O2 transition and how ChR2 modulates selectivity between states is currently unresolved.

Zn(2+) at a cellular crossroads.

Zinc is an essential micronutrient for cellular homeostasis. Initially proposed to only contribute to cellular viability through structural roles and non-redox catalysis, advances in quantifying changes in nM and pM quantities of Zn(2+) have elucidated increasing functions as an important signaling molecule. This includes Zn(2+)-mediated regulation of transcription factors and subsequent protein expression, storage and release of intracellular compartments of zinc quanta into the extracellular space which modulates plasma membrane protein function, as well as intracellular signaling pathways which contribute to the immune response.

Voltage Clamp Fluorometry of P-Type ATPases.

Voltage clamp fluorometry has become a powerful tool to compare partial reactions of P-type ATPases such as the Na(+),K(+)-ATPase and H(+),K(+)-ATPase with conformational dynamics of these ion pumps. Here, we describe the methodology to heterologously express membrane proteins in X. laevis oocytes and site-specifically label these proteins with one or more fluorophores.

A Zinc(II) Photocage Based on a Decarboxylation Metal Ion Release Mechanism for Investigating Homeostasis and Biological Signaling.

Metal ion signaling in biology has been studied extensively with ortho-nitrobenzyl photocages; however, the low quantum yields and other optical properties are not ideal for these applications. We describe the synthesis and characterization of NTAdeCage, the first member in a new class of Zn(2+) photocages that utilizes a light-driven decarboxylation reaction in the metal ion release mechanism. NTAdeCage binds Zn(2+) with sub-pM affinity using a modified nitrilotriacetate chelator and exhibits an almost 6 order of magnitude decrease in metal binding affinity upon uncaging.

Cysteine Substitution and Labeling Provide Insight into Channelrhodopsin-2 Ion Conductance.

Channelrhodopsin-2 is a light-activated cation channel. However, the mechanism of ion conductance is unresolved. Here, we performed cysteine scanning mutagenesis on transmembrane domain 7 followed by labeling with a methanethiosulfonate compound.

Computation and Functional Studies Provide a Model for the Structure of the Zinc Transporter hZIP4.

Members of the Zrt and Irt protein (ZIP) family are a central participant in transition metal homeostasis as they function to increase the cytosolic concentration of zinc and/or iron. However, the lack of a crystal structure hinders elucidation of the molecular mechanism of ZIP proteins. Here, we employed GREMLIN, a co-evolution-based contact prediction approach in conjunction with the Rosetta structure prediction program to construct a structural model of the human (h) ZIP4 transporter.

The large intracellular loop of hZIP4 is an intrinsically disordered zinc binding domain.

The human (h) ZIP4 transporter is a plasma membrane protein which functions to increase the cytosolic concentration of zinc. hZIP4 transports zinc into intestinal cells and therefore has a central role in the absorption of dietary zinc. hZIP4 has eight transmembrane domains and encodes a large intracellular loop between transmembrane domains III and IV, M3M4.

Transmembrane domain three contributes to the ion conductance pathway of channelrhodopsin-2.

Channelrhodopsin-2 (ChR2) is a light-activated nonselective cation channel that is found in the eyespot of the unicellular green alga Chlamydomonas reinhardtii. Despite the wide employment of this protein to control the membrane potential of excitable membranes, the molecular determinants that define the unique ion conductance properties of this protein are not well understood. To elucidate the cation permeability pathway of ion conductance, we performed cysteine scanning mutagenesis of transmembrane domain three followed by labeling with methanethiosulfonate derivatives.

Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel.

The cation selectivity of the ZIP transporters.

Zinc is a required micronutrient for cellular homeostasis and is essential for the structure and/or function of 100s of biological processes. Despite the central importance of zinc in physiology, the mechanism by which this transition metal is transported into cells is not well understood. The first human zinc importer was identified in 2000.

The human ZIP4 transporter has two distinct binding affinities and mediates transport of multiple transition metals.

Zinc is the second most abundant transition metal in the body. Despite the fact that hundreds of biomolecules require zinc for proper function and/or structure, the mechanism of zinc transport into cells is not well-understood. The ZIP (Zrt- and Irt-like proteins; SLC39A) family of proteins acts to increase cytosolic concentrations of zinc.

Examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling.

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels, ion pumps, and transporters. Recent developments have combined site-specific fluorophore labeling alongside TEVC to concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells. We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry.

Ultra light-sensitive and fast neuronal activation with the Ca²+-permeable channelrhodopsin CatCh.

The light-gated cation channel channelrhodopsin-2 (ChR2) has rapidly become an important tool in neuroscience, and its use is being considered in therapeutic interventions. Although wild-type and known variant ChR2s are able to drive light-activated spike trains, their use in potential clinical applications is limited by either low light sensitivity or slow channel kinetics. We present a new variant, calcium translocating channelrhodopsin (CatCh), which mediates an accelerated response time and a voltage response that is ~70-fold more light sensitive than that of wild-type ChR2.

Ligand-dependent effects on the conformational equilibrium of the Na+,K+-ATPase as monitored by voltage clamp fluorometry.

Voltage clamp fluorometry was used to monitor conformational changes associated with electrogenic partial reactions of the Na(+),K(+)-ATPase after changes in the concentration of internal sodium (Na(+)(i)) or external potassium (K(+)(o)). To probe the effects of the Na(+)(i) concentration on the Na(+) branch of the Na(+),K(+)-ATPase, oocytes were depleted of Na(+)(i) and then loaded with external sodium (Na(+)(o)) using the amiloride-sensitive epithelial sodium channel. The K(+) branch of the Na(+),K(+)-ATPase was studied by exposing the oocytes to different K(+)(o) concentrations in the presence and absence of Na(+)(o) to obtain additional information on the apparent affinity for K(+)(o).