Glutamate transporters are involved in direct inhibitory synaptic transmission in the vertebrate retina.

In the central nervous system of vertebrates, glutamate serves as the primary excitatory neurotransmitter. However, in the retina, glutamate released from photoreceptors causes hyperpolarization in post-synaptic ON-bipolar cells through a glutamate-gated chloride current, which seems paradoxical. Our research reveals that this current is modulated by two excitatory glutamate transporters, EAAT5b and EAAT7. In the zebrafish retina, these transporters are located at the dendritic tips of ON-bipolar cells and interact with all four types of cone photoreceptors. The absence of these transporters leads to a decrease in ON-bipolar cell responses, with eaat5b mutants being less severely affected than eaat5b/eaat7 double mutants, which also exhibit altered response kinetics. Biophysical investigations establish that EAAT7 is an active glutamate transporter with a predominant anion conductance. Our study is the first to demonstrate the direct involvement of post-synaptic glutamate transporters in inhibitory direct synaptic transmission at a central nervous system synapse.

Apo state pore opening as functional basis of increased EAAT anion channel activity in episodic ataxia 6.

SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine (P289R EAAT2, P290R EAAT1), have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases.

Mutations associated with epileptic encephalopathy modify EAAT2 anion channel function.

OBJECTIVE: Mutations in the gene solute carrier family member 1A2 (SLC1A2) encoding the excitatory amino acid transporter 2 (EAAT2) are associated with severe forms of epileptic encephalopathy. EAAT2 is expressed in glial cells and presynaptic nerve terminals and represents the main l-glutamate uptake carrier in the mammalian brain. It does not only function as a secondary active glutamate transporter, but also as an anion channel. How naturally occurring mutations affect these two transport functions of EAAT2 and how such alterations cause epilepsy is insufficiently understood. METHODS: Here we studied the functional consequences of three disease-associated mutations, which predict amino acid exchanges p.Gly82Arg (G82R), p.Leu85Pro (L85P), and p.Pro289Arg (P289R), by heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch-clamp recordings of EAAT2 l-glutamate transport and anion current. RESULTS: G82R and L85P exchange amino acid residues that contribute to the formation of the EAAT anion pore. They enlarge the pore diameter sufficiently to permit the passage of l-glutamate and thus function as l-glutamate efflux pathways. The mutation P289R decreases l-glutamate uptake, but increases anion currents despite a lower membrane expression. SIGNIFICANCE: l-glutamate permeability of the EAAT anion pore is an unexpected functional consequence of naturally occurring single amino acid substitutions. l-glutamate efflux through mutant EAAT2 anion channels will cause glutamate excitotoxicity and neuronal hyperexcitability in affected patients. Antagonists that selectively suppress the EAAT anion channel function could serve as therapeutic agents in the future.

Glial Chloride Homeostasis Under Transient Ischemic Stress.

High water permeabilities permit rapid adjustments of glial volume upon changes in external and internal osmolarity, and pathologically altered intracellular chloride concentrations ([Cl-]int) and glial cell swelling are often assumed to represent early events in ischemia, infections, or traumatic brain injury. Experimental data for glial [Cl-]int are lacking for most brain regions, under normal as well as under pathological conditions. We measured [Cl-]int in hippocampal and neocortical astrocytes and in hippocampal radial glia-like (RGL) cells in acute murine brain slices using fluorescence lifetime imaging microscopy with the chloride-sensitive dye MQAE at room temperature. We observed substantial heterogeneity in baseline [Cl-]int, ranging from 14.0 ± 2.0 mM in neocortical astrocytes to 28.4 ± 3.0 mM in dentate gyrus astrocytes. Chloride accumulation by the Na+-K+-2Cl- cotransporter (NKCC1) and chloride outward transport (efflux) through K+-Cl- cotransporters (KCC1 and KCC3) or excitatory amino acid transporter (EAAT) anion channels control [Cl-]int to variable extent in distinct brain regions. In hippocampal astrocytes, blocking NKCC1 decreased [Cl-]int, whereas KCC or EAAT anion channel inhibition had little effect. In contrast, neocortical astrocytic or RGL [Cl-]int was very sensitive to block of chloride outward transport, but not to NKCC1 inhibition. Mathematical modeling demonstrated that higher numbers of NKCC1 and KCC transporters can account for lower [Cl-]int in neocortical than in hippocampal astrocytes. Energy depletion mimicking ischemia for up to 10 min did not result in pronounced changes in [Cl-]int in any of the tested glial cell types. However, [Cl-]int changes occurred under ischemic conditions after blocking selected anion transporters. We conclude that stimulated chloride accumulation and chloride efflux compensate for each other and prevent glial swelling under transient energy deprivation.

Cellular Physiology and Pathophysiology of EAAT Anion Channels.

Excitatory amino acid transporters (EAATs) optimize the temporal resolution and energy demand of mammalian excitatory synapses by quickly removing glutamate from the synaptic cleft into surrounding neuronal and glial cells and ensuring low resting glutamate concentrations. In addition to secondary active glutamate transport, EAATs also function as anion channels. The channel function of these transporters is conserved in all homologs ranging from archaebacteria to mammals; however, its physiological roles are insufficiently understood. There are five human EAATs, which differ in their glutamate transport rates. Until recently the high-capacity transporters EAAT1, EAAT2, and EAAT3 were believed to conduct only negligible anion currents, with no obvious function in cell physiology. In contrast, the low-capacity glutamate transporters EAAT4 and EAAT5 are thought to regulate neuronal signaling as glutamate-gated channels. In recent years, new experimental approaches and novel animal models, together with the discovery of a human genetic disease caused by gain-of-function mutations in EAAT anion channels have enabled identification of the first physiological and pathophysiological roles of EAAT anion channels.

Increased glutamate transporter-associated anion currents cause glial apoptosis in episodic ataxia 6.

Episodic ataxia type 6 is an inherited neurological condition characterized by combined ataxia and epilepsy. A severe form of this disease with episodes combining ataxia, epilepsy and hemiplegia was recently associated with a proline to arginine substitution at position 290 of the excitatory amino acid transporter 1 in a heterozygous patient. The excitatory amino acid transporter 1 is the predominant glial glutamate transporter in the cerebellum. However, this glutamate transporter also functions as an anion channel and earlier work in heterologous expression systems demonstrated that the mutation impairs the glutamate transport rate, while increasing channel activity. To understand how these changes cause ataxia, we developed a constitutive transgenic mouse model. Transgenic mice display epilepsy, ataxia and cerebellar atrophy and, thus, closely resemble the human disease. We observed increased glutamate-activated chloride efflux in Bergmann glia that triggers the apoptosis of these cells during infancy. The loss of Bergmann glia results in reduced glutamate uptake and impaired neural network formation in the cerebellar cortex. This study shows how gain-of-function of glutamate transporter-associated anion channels causes ataxia through modifying cerebellar development.

Functional consequences of SLC1A3 mutations associated with episodic ataxia 6.

The episodic ataxias (EA) are a group of inherited neurological diseases characterized by paroxysmal cerebellar incoordination. There exist nine forms of episodic ataxia with distinct neurological symptoms and genetic origins. Episodic ataxia type 6 (EA6) differs from other EA forms in long attack duration, epilepsy and absent myokymia, nystagmus, and tinnitus. It has been described in seven families, and mutations in SLC1A3, the gene encoding the glial glutamate transporter EAAT1, were reported in each family. How these mutations affect EAAT1 expression, subcellular localization, and function, and how such alterations result in the complex neurological phenotype of EA6 is insufficiently understood. We here compare the functional consequences of all currently known mutations by heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch clamp recordings of EAAT1 transport and anion currents. We observed impairments of multiple EAAT1 properties ranging from changes in transport function, impaired trafficking to increased protein expression. Many mutations caused only slight changes illustrating how sensitively the cerebellum reacts on impaired EAAT1 functions.

Impaired K+ binding to glial glutamate transporter EAAT1 in migraine.

SLC1A3 encodes the glial glutamate transporter hEAAT1, which removes glutamate from the synaptic cleft via stoichiometrically coupled Na+-K+-H+-glutamate transport. In a young man with migraine with aura including hemiplegia, we identified a novel SLC1A3 mutation that predicts the substitution of a conserved threonine by proline at position 387 (T387P) in hEAAT1. To evaluate the functional effects of the novel variant, we expressed the wildtype or mutant hEAAT1 in mammalian cells and performed whole-cell patch clamp, fast substrate application, and biochemical analyses. T387P diminishes hEAAT1 glutamate uptake rates and reduces the number of hEAAT1 in the surface membrane. Whereas hEAAT1 anion currents display normal ligand and voltage dependence in cells internally dialyzed with Na+-based solution, no anion currents were observed with internal K+. Fast substrate application demonstrated that T387P abolishes K+-bound retranslocation. Our finding expands the phenotypic spectrum of genetic variation in SLC1A3 and highlights impaired K+ binding to hEAAT1 as a novel mechanism of glutamate transport dysfunction in human disease.

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation.

Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride-sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl- ]int to be controlled by two opposing transport processes: chloride is actively accumulated by the Na+ -K+ -2Cl- cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl- ]int to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion-selective channels during glutamate transport, and thus represent a class of ligand-gated anion channels. Age-dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT-1 increased [Cl- ]int in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl- ]int . Other tested chloride channels or chloride transporters do not contribute to [Cl- ]int under our experimental conditions. Neither genetic removal of ClC-2 nor pharmacological block of K+ -Cl- cotransporter change resting Bergmann glial [Cl- ]int in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388-400.

CNS Schwann cells display oligodendrocyte precursor-like potassium channel activation and antigenic expression in vitro.

Central nervous system (CNS) injury triggers production of myelinating Schwann cells from endogenous oligodendrocyte precursors (OLPs). These CNS Schwann cells may be attractive candidates for novel therapeutic strategies aiming to promote endogenous CNS repair. However, CNS Schwann cells have been so far mainly characterized in situ regarding morphology and marker expression, and it has remained enigmatic whether they display functional properties distinct from peripheral nervous system (PNS) Schwann cells. Potassium channels (K+) have been implicated in progenitor and glial cell proliferation after injury and may, therefore, represent a suitable pharmacological target. In the present study, we focused on the function and expression of voltage-gated K+ channels Kv(1-12) and accessory ß-subunits in purified adult canine CNS and PNS Schwann cell cultures using electrophysiology and microarray analysis and characterized their antigenic phenotype. We show here that K+ channels differed significantly in both cell types. While CNS Schwann cells displayed prominent K D-mediated K+ currents, PNS Schwann cells elicited K(D-) and K(A-type) K+ currents. Inhibition of K+ currents by TEA and Ba2+ was more effective in CNS Schwann cells. These functional differences were not paralleled by differential mRNA expression of Kv(1-12) and accessory ß-subunits. However, O4/A2B5 and GFAP expressions were significantly higher and lower, respectively, in CNS than in PNS Schwann cells. Taken together, this is the first evidence that CNS Schwann cells display specific properties not shared by their peripheral counterpart. Both Kv currents and increased O4/A2B5 expression were reminiscent of OLPs suggesting that CNS Schwann cells retain OLP features during maturation.

Mutating a conserved proline residue within the trimerization domain modifies Na+ binding to excitatory amino acid transporters and associated conformational changes.

Excitatory amino acid transporters (EAATs) are crucial for glutamate homeostasis in the mammalian central nervous system. They are not only secondary active glutamate transporters but also function as anion channels, and different EAATs vary considerably in glutamate transport rates and associated anion current amplitudes. A naturally occurring mutation, which was identified in a patient with episodic ataxia type 6 and that predicts the substitution of a highly conserved proline at position 290 by arginine (P290R), was recently shown to reduce glutamate uptake and to increase anion conduction by hEAAT1. We here used voltage clamp fluorometry to define how the homologous P259R mutation modifies the functional properties of hEAAT3. P259R inverts the voltage dependence, changes the sodium dependence, and alters the time dependence of hEAAT3 fluorescence signals. Kinetic analysis of fluorescence signals indicate that P259R decelerates a conformational change associated with sodium binding to the glutamate-free mutant transporters. This alteration in the glutamate uptake cycle accounts for the experimentally observed changes in glutamate transport and anion conduction by P259R hEAAT3.

A point mutation associated with episodic ataxia 6 increases glutamate transporter anion currents.

Episodic ataxia is a human genetic disease characterized by paroxysmal cerebellar incoordination. There are several genetically and clinically distinct forms of this disease, and one of them, episodic ataxia type 6, is caused by mutations in the gene encoding a glial glutamate transporter, the excitatory amino acid transporter-1. So far, reduced glutamate uptake by mutant excitatory amino acid transporter-1 has been thought to be the main pathophysiological process in episodic ataxia type 6. However, excitatory amino acid transporter-1 does not only mediate secondary-active glutamate transport, but also functions as an ion channel. Here, we examined the effects of a disease-associated point mutation, P290R, on glutamate transport, anion current as well as on the subcellular distribution of excitatory amino acid transporter-1 using heterologous expression in mammalian cells. P290R reduces the number of excitatory amino acid transporter-1 in the surface membrane and impairs excitatory amino acid transporter-1-mediated glutamate uptake. Cells expressing P290R excitatory amino acid transporter-1 exhibit larger anion currents than wild-type cells in the absence as well as in the presence of external l-glutamate, despite a lower number of mutant transporters in the surface membrane. Noise analysis revealed unaltered unitary current amplitudes, indicating that P290R modifies opening and closing, and not anion permeation through mutant excitatory amino acid transporter-1 anion channels. These findings identify gain-of-function of excitatory amino acid transporter anion conduction as a pathological process in episodic ataxia. Episodic ataxia type 6 represents the first human disease found to be associated with altered function of excitatory amino acid transporter anion channels and illustrates possible physiological and pathophysiological impacts of this functional mode of this class of glutamate transporters.

Noise analysis to study unitary properties of transporter-associated ion channels.

Excitatory amino acid transporters (EAATs) do not only mediate secondary-active glutamate uptake but also function as anion channels. We recently used macroscopic current recordings and noise analysis to determine unitary current amplitudes of anion channels associated with a neuronal EAAT isoform, EAAT4. We found that, at symmetrical NO(3)(-), EAAT4 anion channels exhibit a single channel conductance of ~1 pS in the absence as well as in the presence of glutamate. These results indicate that glutamate increases EAAT4 anion currents by modifying exclusively open probabilities, however, leaves unitary current amplitudes unaffected. Noise analysis has been developed for ion channels with a single conductance state and limitations might ensue when using this approach for transporter-associated ion channels. We here performed stochastic simulations of EAAT transporter-associated anion channels and noise analysis of simulated currents to assess the reliability and possible limitations of this technique in studying this special class of ion channels.

Substrate-dependent gating of anion channels associated with excitatory amino acid transporter 4.

EAAT glutamate transporters do not only function as secondary-active glutamate transporters but also as anion channels. EAAT anion channel activity depends on transport substrates. For most isoforms, it is negligible without external Na(+) and increased by external glutamate. We here investigated gating of EAAT4 anion channels with various cations and amino acid substrates using patch clamp experiments on a mammalian cell line. We demonstrate that Li(+) can substitute for Na(+) in supporting substrate-activated anion currents, albeit with changed voltage dependence. Anion currents were recorded in glutamate, aspartate, and cysteine, and distinct time and voltage dependences were observed. For each substrate, gating was different in external Na(+) or Li(+). All features of voltage-dependent and substrate-specific anion channel gating can be described by a simplified nine-state model of the transport cycle in which only amino acid substrate-bound states assume high anion channel open probabilities. The kinetic scheme suggests that the substrate dependence of channel gating is exclusively caused by differences in substrate association and translocation. Moreover, the voltage dependence of anion channel gating arises predominantly from electrogenic cation binding and membrane translocation of the transporter. We conclude that all voltage- and substrate-dependent conformational changes of the EAAT4 anion channel are linked to transitions within the transport cycle.

Malate transport by the vacuolar AtALMT6 channel in guard cells is subject to multiple regulation.

Gas exchange in plants is controlled by guard cells, specialized cells acting as turgor pressure-driven valves. Malate is one of the major anions accumulated inside the vacuole during stomatal opening counteracting the positive charge of potassium. AtALMT6, a member of the aluminum-activated malate transporter family, is expressed in guard cells of leaves and stems as well as in flower organs of Arabidopsis thaliana. An AtALMT6-GFP fusion protein was targeted to the vacuolar membrane both in transient and stable expression systems. Patch-clamp experiments on vacuoles isolated from AtALMT6-GFP over-expressing Arabidopsis plants revealed large inward-rectifying malate currents only in the presence of micromolar cytosolic calcium concentrations. Further analyses showed that vacuolar pH and cytosolic malate regulate the threshold of activation of AtALMT6-mediated currents. The interplay of these two factors determines the AtALMT6 function as a malate influx or efflux channel depending on the tonoplast potential. Guard cell vacuoles isolated from Atalmt6 knock-out plants displayed reduced malate currents compared with wild-type vacuoles. This reduction, however, was not accompanied by phenotypic differences in the stomatal movements in knock-out plants, probably because of functional redundancy of malate transporters in guard cell vacuoles.

Hetero-oligomerization of neuronal glutamate transporters.

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo- and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.

A conserved aspartate determines pore properties of anion channels associated with excitatory amino acid transporter 4 (EAAT4).

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp(112)) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.

The Arabidopsis vacuolar malate channel is a member of the ALMT family.

In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.

Tim50 maintains the permeability barrier of the mitochondrial inner membrane.

Transport of metabolites across the mitochondrial inner membrane is highly selective, thereby maintaining the electrochemical proton gradient that functions as the main driving force for cellular adenosine triphosphate synthesis. Mitochondria import many preproteins via the presequence translocase of the inner membrane. However, the reconstituted Tim23 protein constitutes a pore remaining mainly in its open form, a state that would be deleterious in organello. We found that the intermembrane space domain of Tim50 induced the Tim23 channel to close. Presequences overcame this effect and activated the channel for translocation. Thus, the hydrophilic cis domain of Tim50 maintains the permeability barrier of mitochondria by closing the translocation pore in a presequence-regulated manner.

Protein insertion into the mitochondrial inner membrane by a twin-pore translocase.

The mitochondrial inner membrane imports numerous proteins that span it multiple times using the membrane potential Deltapsi as the only external energy source. We purified the protein insertion complex (TIM22 complex), a twin-pore translocase that mediated the insertion of precursor proteins in a three-step process. After the precursor is tethered to the translocase without losing energy from the Deltapsi, two energy-requiring steps were needed. First, Deltapsi acted on the precursor protein and promoted its docking in the translocase complex. Then, Deltapsi and an internal signal peptide together induced rapid gating transitions in one pore and closing of the other pore and drove membrane insertion to completion. Thus, protein insertion was driven by the coordinated action of a twin-pore complex in two voltage-dependent steps.