Robert E. Dempski

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. CatChs superior properties stem from its enhanced Ca2+ permeability. An increase in [Ca2+]i elevates the internal surface potential, facilitating activation of voltage-gated Na+ channels and indirectly increasing light sensitivity. Repolarization following light-stimulation is markedly accelerated by Ca2+-dependent BK channel activation. Our results demonstrate a previously unknown principle: shifting permeability from monovalent to divalent cations to increase sensitivity without compromising fast kinetics of neuronal activation. This paves the way for clinical use of light-gated channels.

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. Mutations in one member of the ZIP family of proteins, the human ZIP4 (hZIP4; SLC39A4) protein, can result in the disease acrodermatitis enteropathica (AE). AE is characterized by growth retardation and diarrhea, as well as behavioral and neurological disturbances. While the cellular distribution of hZIP4 protein expression has been elucidated, the cation specificity, kinetic parameters of zinc transport, and residues involved in cation translocation are unresolved questions. Therefore, we have established a high signal-to-noise zinc uptake assay following heterologous expression of hZIP4 in Xenopus laevis oocytes. The results from our experiments have demonstrated that zinc, copper(II), and nickel can be transported by hZIP4 when the cation concentration is in the micromolar range. We have also identified a nanomolar binding affinity where copper(II) and zinc can be transported. In contrast, under these conditions, nickel can bind but is not transported by hZIP4. Finally, labeling of hZIP4 with maleimide or diethylpyrocarbonate indicates that extracellularly accessible histidine, but not cysteine, residues are required, either directly or indirectly, for cation uptake. The results of our experiments identify at least two coordination sites for divalent cations and provide a new framework for investigating the ZIP family of proteins.

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

Background: ZIP transporters increase the cytosolic concentration of first row transition metals. Results: We have developed a structural model of hZIP4 by combining protein prediction methods with in situ experiments. Conclusion: Analysis of our experiments provides insight into the permeation pathway of hZIP4. Significance: Comparison of this model to membrane transporter crystal structures provides a structural linkage to MFS proteins. 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 predicted contact data are best fit by modeling hZIP4 as a dimer. Mutagenesis of residues that comprise a central putative hZIP4 transmembrane transition metal coordination site in the structural model alter the kinetics and specificity of hZIP4. Comparison of the hZIP4 dimer model to all known membrane protein structures identifies the 12-transmembrane monomeric Piriformospora indica phosphate transporter (PiPT), a member of the major facilitator superfamily (MFS), as a likely structural homolog.

Functional Significance of E2 State Stabilization by Specific α/β-Subunit Interactions of Na,K- and H,K-ATPase

The β-subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic α-subunit but also modulate the transport activity of the holoenzymes. In this study, we show that tryptophan replacement of two highly conserved tyrosines in the transmembrane domain of both Na,K- and gastric H,K-ATPase β-subunits resulted in considerable shifts of the voltage-dependent E1P/E2P distributions toward the E1P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimide-labeled ATPases. The shifts in conformational equilibria were accompanied by significant decreases in the apparent affinities for extracellular K+ that were moderate for the Na,K-ATPase β-(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase β-(Y44W,Y48W) variant. Moreover in the Na,K-ATPase β-(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na+ and the subsequent E2P-E1P conversion, as determined from transient current kinetics, was significantly accelerated, resulting in enhanced Na+ competition for extracellular K+ binding especially at extremely negative potentials. Analogously the reverse binding of extracellular protons and subsequent E2P-E1P conversion was accelerated by the H,K-ATPase β-(Y44W,Y48W) mutation, and H+ secretion was strongly impaired. Remarkably tryptophan replacements of residues in the M7 segment of Na,K- and H,K-ATPase α-subunits, which are at interacting distance to the β-tyrosines, resulted in similar E1 shifts, indicating their participation in stabilization of E2. Thus, interactions between selected residues within the transmembrane regions of α- and β-subunits of P2C-type ATPases exert an E2-stabilizing effect, which is of particular importance for efficient H+ pumping by H,K-ATPase under in vivo conditions.

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. Since that time, a new family of proteins, named ZIP, for Zrt-, Irt-like proteins, has been shown to be expressed in a tissue-specific manner in humans. There are a total of 14 members of this family, which can be further divided into four subfamilies based on sequence similarities: ZIPI, ZIPII, gufA and LIV-1. It has been shown that these proteins are expressed on the plasma membrane as well as on intracellular organelles and each function or are proposed to function to increase the cytosolic concentration of zinc. While the subcellular localization of most of the ZIP family of proteins has been elucidated, the mechanistic details of these proteins including the driving force of cation translocation, residues essential for transport and the molecular determinants which define the differing cation selectivity between the ZIP family of proteins have not been well resolved. The objective of this review is to describe the current understanding of cation transport, mediated by the mammalian family of ZIP proteins and to present some molecular determinants that may contribute to the differing substrate specificity for this family of proteins.

Fluorometric Measurements of Intermolecular Distances between the α- and β-Subunits of the Na+/K+-ATPase

The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the α-subunit, which mediates ATP hydrolysis and ion translocation, as well as the β-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the α- and β-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the α- and β-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the α-subunit and the β-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the β-subunit in relation to the α-subunit and suggest that the α- and β-subunits move toward each other during the E2 to E1 conformational transition.

The β Subunit of the Na+/K+-ATPase Follows the Conformational State of the Holoenzyme

The Na+/K+-ATPase is a ubiquitous plasma membrane ion pump that utilizes ATP hydrolysis to regulate the intracellular concentration of Na+ and K+. It is comprised of at least two subunits, a large catalytic α subunit that mediates ATP hydrolysis and ion transport, and an ancillary β subunit that is required for proper trafficking of the holoenzyme. Although processes mediated by the α subunit have been extensively studied, little is known about the participation of the β subunit in conformational changes of the enzyme. To elucidate the role of the β subunit during ion transport, extracellular amino acids proximal to the transmembrane region of the sheep β1 subunit were individually replaced for cysteines. This enabled sulfhydryl-specific labeling with the environmentally sensitive fluorescent dye tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes. Investigation by voltage-clamp fluorometry identified three reporter positions on the β1 subunit that responded with fluorescence changes to alterations in ionic conditions and/or membrane potential. These experiments for the first time show real-time detection of conformational rearrangements of the Na+/K+-ATPase through a fluorophore-labeled β subunit. Simultaneous recording of presteady-state or stationary currents together with fluorescence signals enabled correlation of the observed environmental changes of the β subunit to certain reaction steps of the Na+/K+-ATPase, which involve changes in the occupancy of the two principle conformational states, E1P and E2P. From these experiments, evidence is provided that the β1-S62C mutant can be directly used to monitor the conformational state of the enzyme, while the F64C mutant reveals a relaxation process that is triggered by sodium transport but evolves on a much slower time scale. Finally, shifts in voltage dependence and kinetics observed for mutant K65C show that this charged lysine residue, which is conserved in β1 isoforms, directly influences the effective potential that determines voltage dependence of extracellular cation binding and release.

Voltage clamp fluorometry: Combining fluorescence and electrophysiological methods to examine the structure–function of the Na+/K+-ATPase

This paper summarizes our recent work investigating the conformational dynamics and structural arrangement of the Na(+)/K(+)-ATPase using voltage clamp fluorometry as well as the latest biochemical, biophysical and structural results from other laboratories. Our research has been focused on combining site-specific fluorophore labeling on the alpha, beta and/or gamma subunit with electrophysiological studies to investigate partial reactions of the ion pump by monitoring changes in fluorescence intensity following voltage pulses and/or solution exchange. As a consequence of these studies, we have been able to identify a residue on the beta subunit, which following labeling with tetramethylrhodamine-6-maleimide can be used as a reporter group to monitor the conformational state of the holoenzyme. Furthermore, we have been able to delineate distance constraints between the alpha, beta and gamma subunits and to examine the relative movements of these proteins during ion transport. Concurrent to this research, significant advancements have been made in understanding the molecular mechanism of the Na(+)/K(+)-ATPase. Thus, our research will be compared with the results from other groups and future experimental directions will be proposed.

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. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2.

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. This review highlights some recent advances in our understanding of zinc signaling.