Biology subjects

Fahlke, C.

Publications and source records attributed to Fahlke, C..

8 recordsLinked to original sources

bioRxiv2026

Evolutionary optimization of allosteric activation by Cl- and Cl- conduction in vesicular glutamate transporters

Vesicular glutamate transporters harness proton gradients to load glutamate into synaptic vesicles, while mediating luminal chloride efflux through a channel-like conductance. We combined heterologous expression in mammalian cells, whole-cell patch-clamp recordings and mathematical modeling to functionally characterize the Drosophila melanogaster vesicular glutamate transporter DVGLUT and to compare it to a rat counterpart, rVGLUT1. As in mammalian VGLUTs, DVGLUT glutamate transport is coupled to proton exchange, in a 1:1 stoichiometry. In both, luminal Cl- is necessary as allosteric activator, however, the allosteric affinity is higher in fly than in rat transporters. DVGLUT anion channels exhibit lower unitary currents, but higher anion channel open probabilities, resulting in larger Cl- currents for the fly transporter in the presence of cytoplasmic glutamate. The higher allosteric affinity together with the enhanced anion channel activity may serve as evolutionary adaptation of VGLUTs to lower ion concentrations in Drosophila, illustrating the impact of these particular features for synaptic vesicle filling.

neuroscience↗

bioRxiv2025

SLC26A11 is an atypical solute carrier with dual transport-channel function mediating lysosomal sulfate transport

Membrane transporters and channels are generally assumed to be based on distinct structural and functional principles. SLC26A11, a solute carrier with high expression levels in the brain, has been proposed to function as either an anion transporter or a channel. Here, we resolve this apparent discrepancy by demonstrating that SLC26A11 is a dual-function protein capable of operating as both a sulfate transporter and a chloride channel. By resolving its structure and combining biochemical studies and molecular dynamics simulations, we show that SLC26A11 exhibits all the hallmarks of a secondary transporter. The mechanistic basis for its selective ion transport identifies the protein as the elusive lysosomal sulfate exporter. Additionally, we demonstrate that SLC26A11 exhibits an uncoupled, channel-like chloride conductance gated by proton:sulfate symport. Our finding that the chloride-conducting state arises from the transport cycle may contribute to the development of novel therapeutic strategies for treating brain edema, and the identification of its role in lysosome sulfate efflux may provide new approaches to study and treat lysosomal storage diseases.

biochemistry↗

bioRxiv2024

Allosteric modulation of proton binding confers Cl- activation and glutamate selectivity to vesicular glutamate transporters

Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and remove luminal Cl- via an additional anion channel mode. Both of these transport functions are stimulated by luminal acidification, luminal-positive membrane potential, and luminal Cl-. We studied VGLUT1 transporter/channel activation using a combination of heterologous expression, cellular electrophysiology, fast solution exchange, and mathematical modeling. Cl- channel gating can be described with a kinetic scheme that includes two protonation sites and distinct opening, closing, and Cl--binding rates for each protonation state. Cl- binding promotes channel opening by modifying the pKa values of the protonation sites and rates of pore opening and closure. VGLUT1 transports glutamate and aspartate at distinct stoichiometries: H+-glutamate exchange at 1:1 stoichiometry and aspartate uniport. Neurotransmitter transport with variable stoichiometry can be described with an alternating access model that assumes that transporters without substrate translocate in the doubly protonated state to the inward-facing conformation and return with the bound amino acid substrate as either singly or doubly protonated. Glutamate, but not aspartate, promotes the release of one proton from inward-facing VGLUT1, resulting in preferential H+-coupled glutamate exchange. Cl- stimulates glutamate transport by making the glutamate-binding site accessible to cytoplasmic glutamate and by facilitating transitions to the inward-facing conformation after outward substrate release. We conclude that allosteric modification of transporter protonation by Cl- is crucial for both VGLUT1 transport functions.

biophysics↗

bioRxiv2024

Oligomerization and cellular localization of SLC26A11

The solute carrier family 26 (SLC26) encompasses multifunctional anion exchangers in all kingdoms of life. SLC26 proteins are known to assemble as dimers, and co-expression of multiple isoforms in certain cells raises the question whether different SLC26s can assemble into hetero-dimers. We focused on SLC26A11, a broadly expressed isoform that differs from other isoforms in its subcellular localization. Whereas the vast majority of SLC26-FP fusion proteins, i.e. SLC26A1, SLC26A2, SlC26A3, SLC26A4/pendrin, SLC26A5/prestin, SLC26A6, SlC26A7, and SLC26A9, localize to the surface membrane of transfected mammalian cells, we found exclusive lysosomal localization of SLC26A11. Renal collecting duct intercalated cells express SLC26A11 together with SLC26A4/pendrin and SLC26A7, and we therefore tested whether heterodimerization between these transporters might result in SLC26 transporter re-localization. Neither in HEK293T nor in immortalized intercalated cells co-expressing SLC26A11 with SLC26A4/pendrin or with SLC26A7, changes of SLC26A11 localization were observed. Moreover, native gel electrophoresis did not provide any evidence for heterodimerization of these isoforms. We next tested heterodimerization of SLC26A11 with SLC26A1, SLC26A2, SLC26A6 or SLC26A9 via co-expression in HEK293T cells and confocal imaging. For all combinations, no changes in subcellular distribution were observed. We conclude that SLC26A11 does not heterodimerize with other SLC26 proteins, and that heterodimerization does not target SLC26A11 to cellular surface membranes.

cell biology↗

bioRxiv2024

Transport mechanism of DgoT, a bacterial homolog of SLC17 organic anion transporters

The solute carrier 17 (SLC17) family contains anion transporters that accumulate neuro-transmitters in secretory vesicles, remove carboxylated monosaccharides from lysosomes, or extrude organic anions from the kidneys and the liver. We combined classical molecular dynamics simulations, Markov state modeling and hybrid first principles quantum mechani-cal/classical mechanical (QM/MM) simulations with experimental approaches to describe the transport mechanisms of a model bacterial protein, the D-galactonate transporter DgoT, at atomic resolution. We found that protonation of D46 and E133 precedes galactonate binding and that substrate binding induces closure of the extracellular gate, with the conserved R47 coupling substrate binding to transmembrane helix movement. After isomerization to an inward-facing conformation, deprotonation of E133 and subsequent proton transfer from D46 to E133 opens the intracellular gate and permits galactonate dissociation either in its unprotonated form or after proton transfer from E133. After release of the second proton, apo DgoT returns to the outward-facing conformation. Our results provide a framework to understand how various SLC17 transport functions with distinct transport stoichiometries can be attained through subtle variations in proton and substrate binding/unbinding.

biophysics↗

bioRxiv2021

Determination of protein stoichiometries via dual-color colocalization with single molecule localization microscopy

The oligomeric state of plasma membrane proteins is the result of the interactions between individual proteins and an important determinant of their function. Most approaches used to address this question rely on extracting these complexes from their native environment, which may disrupt weaker interactions. Therefore, microscopy techniques have been increasingly used in recent years to determine oligomeric states in situ. Classical light microscopy suffers from insufficient resolution, but super-resolution methods such as single molecule localization microscopy (SMLM) can circumvent this problem. When using SMLM to determine oligomeric states of proteins, subunits are labeled with fluorescent proteins that only emit light following activation or conversion at different wavelengths. Typically, individual molecules are counted based on a binomial distribution analysis of emission events detected within the same diffraction-limited volume. This strategy requires low background noise, a high recall rate for the fluorescent tag and intensive post-imaging data processing. To overcome these limitations, we developed a new method based on SMLM to determine the oligomeric state of plasma membrane proteins. Our dual-color colocalization (DCC) approach allows for accurate in situ counting even with low efficiencies of fluorescent protein detection. In addition, it is robust in the presence of background signals and does not require temporal clustering of localizations from individual proteins within the same diffraction limited volume, which greatly simplifies data acquisition and processing. We used DCC-SMLM to resolve the controversy surrounding the oligomeric state of two SLC26 multifunctional anion exchangers and to determine the oligomeric state of four members of the SLC17 family of organic anion transporters.

cell biology↗

bioRxiv2020

An amino-terminal point mutation increases EAAT2 anion currents without affecting glutamate transport rates

Excitatory amino acid transporters (EAATs) are prototypic dual function proteins that function as coupled glutamate/Na+/H+/K+ transporters and as anion-selective channels. Both transport functions are intimately intertwined at the structural level: secondary active glutamate transport is based on elevator-like movements of the mobile transport domain across the membrane, and the lateral movement of this domain results in anion channel opening. This particular anion channel gating mechanism predicts the existence of mutant transporters with changed anion channel properties, but without alteration in glutamate transport. We here report that the L46P mutation in the human EAAT2 transporter fulfils this prediction. L46 is a pore-forming residue of the EAAT2 anion channels at the cytoplasmic entrance into the ion conduction pathway. In whole-cell patch clamp recordings, we observed larger macroscopic anion current amplitudes for L46P than for WT EAAT2. Moreover, changes in selectivity made gluconate permeant in L46P EAAT2. Non-stationary noise analysis revealed unchanged unitary current amplitudes in mutant EAAT2 anion channels. Rapid L-glutamate application under forward transport conditions demonstrated that L46P does not reduce the transport rate of individual transporters. Since individual transport rates and unitary anion current amplitudes are unaffected, absolute open probabilities of EAAT2 anion channels were quantified as ratio of anion currents by glutamate uptake currents. We found up to sevenfold increased absolute open probability of L46P EAAT2 anion channels. Our results reveal an important determinant of the diameter of EAAT2 anion pore and demonstrate the existence of anion channel gating processes outside the EAAT uptake cycle.

physiology↗

bioRxiv2019

Metabolic Energy Sensing by Mammalian CLC Anion/Proton Exchangers

Mammalian CLC anion/proton exchangers control the pH and [Cl-] of the endolysosomal system, one of the major cellular nutrient uptake pathways. We explored the regulation of the vesicular transporters ClC-3, ClC-4, and ClC-5 by the adenylic system components ATP, ADP, and AMP. Using heterologous expression and whole-cell electrophysiology, we demonstrated that cytosolic ATP and ADP but not AMP and Mg2+-free ADP enhance CLC ion transport via binding to the protein C-terminal CBS domains. Biophysical investigations revealed that the effects depend on the delivery of intracellular protons into the CLC transport machinery and result from modified voltage-dependence and altered probability that CLC proteins undergo silent non-transporting cycles. Our findings demonstrate that the CLC CBS domains are able to serve as energy sensors by detecting changes in the cytosolic ATP/ADP/AMP equilibrium. The adenine nucleotide regulation of vesicular Cl-/H+ exchange creates a link between the activity of the endolysosomal system and the cellular metabolic state.

cell biology↗