Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K ϩ or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

The molecular identity of K ؉ channels involved in Ehrlich cell volume regulation is unknown. A background K ؉ conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K ؉ channels)-type of two pore-domain K ؉ channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K ؉ currents in the absence intracellular Ca 2؉ . Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK a in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of Potassium channels are multimeric membrane proteins capable of allowing the passage of K ϩ ions across the membrane down their electrochemical potential gradient. Their functions range from the propagation of the action potential and the control of excitability to transepithelial transport and the homeostasis of cell volume. There are many varieties of K ϩ channels distinguishable by their functional properties and pharmacological sensitivities. From the molecular point of view, three major families have been distinguished (1): voltage-gated K V channels, Kir inward rectifiers and SK Ca /IK Ca Ca 2ϩ -dependent K ϩ channels. These previously described K ϩ channels have only one pore domain (P) and form tetramers with each monomer contributing one P domain to the selectivity filter.A novel family of K ϩ channels which, exceptionally, have two P regions in tandem and four putative transmembrane helices (2P-4TM

Excitatory synaptic transmission stimulates brain tissue glycolysis. This phenomenon is the signal detected in FDG-PET imaging and, through enhanced lactate production, is also thought to contribute to the fMRI signal. Using a method based on Förster resonance energy transfer in mouse astrocytes, we have recently observed that a small rise in extracellular K+ can stimulate glycolysis by over 300% within seconds. The K+ response was blocked by ouabain, but intracellular engagement of the Na+/K+ ATPase pump with Na+ was ineffective, suggesting that the canonical feedback regulatory pathway involving the Na+ pump and ATP depletion is only permissive and that a second mechanism is involved. Because of their predominant K+ permeability and high expression of the electrogenic Na+/HCO3− cotransporter NBCe1, astrocytes respond to a rise in extracellular K+ with plasma membrane depolarization and intracellular alkalinization. In the present article we show that a fast glycolytic response can be elicited independently of K+ by plasma membrane depolarization or by intracellular alkalinization. The glycolytic response to K+ was absent in astrocytes from NBCe1 null mice (Slc4a4) and was blocked by functional or pharmacological inhibition of the NBCe1. Hippocampal neurons acquired K+-sensitive glycolysis upon heterologous NBCe1 expression. The phenomenon could also be reconstituted in HEK293 cells by co-expression of the NBCe1 and a constitutively-open K+ channel. We conclude that the NBCe1 is a key element in a feedforward mechanism linking excitatory synaptic transmission to fast modulation of glycolysis in astrocytes.

Potassium channels share a common selectivity filter that determines the conduction characteristics of the pore. Diversity in K ؉ channels is given by how they are gated open. TASK-2, TALK-1, and TALK-2 are two-pore region (2P) KCNK K ؉ channels gated open by extracellular alkalinization. We have explored the mechanism for this alkalinization-dependent gating using molecular simulation and site-directed mutagenesis followed by functional assay. We show that the side chain of a single arginine residue (R224) near the pore senses pH in TASK-2 with an unusual pK a of 8.0, a shift likely due to its hydrophobic environment. R224 would block the channel through an electrostatic effect on the pore, a situation relieved by its deprotonation by alkalinization. A lysine residue in TALK-2 fulfills the same role but with a largely unchanged pK a, which correlates with an environment that stabilizes its positive charge. In addition to suggesting unified alkaline pH-gating mechanisms within the TALK subfamily of channels, our results illustrate in a physiological context the principle that hydrophobic environment can drastically modulate the pK a of charged amino acids within a protein.KCNK channels ͉ molecular simulation ͉ TALK-2 ͉ TASK-2 A ll K ϩ channels contain a highly conserved sequence, the P domain, which forms the selectivity filter and generally six transmembrane ␣-helices. The K ϩ channel pore is formed by four identical subunits, each comprising a P-domain and two of the six transmembrane ␣-helices encircling the ion conduction pathway with a 4-fold symmetry. Structures attached to the pore-forming domains are able to transduce signals, such as changes in transmembrane voltage and intra-or extracellular messages, into gating of the pore (1, 2). Potassium channels of the KCNK superfamily (3, 4) are remarkable in that they possess two P-domains and four ␣-helices in each subunit, form dimers, and are mostly open (''leak channels'') at resting potential. Potassium-selective leaks are fundamental to the function of various cells including nerve, muscle, and epithelia. There are 16 mammalian members to the KCNK family and their gating is variously regulated by free fatty acids, membrane tension, G protein-generated signaling, and extracellular pH. Among KCNK channels gated by extracellular pH, TASK-1 and TASK-3 form a subfamily (TASK) of channels blocked by extracellular protons (5-8). A second subfamily (TALK) of KCNK channels comprises TASK-2, TALK-1, and TALK-2 ¶ , all activated by extracellular alkalinization. TASK-2 participates in ion fluxes necessary for cell volume regulation (10, 11), and its physiological and possible pathological importance has also been highlighted by studies in a TASK-2 knockout mouse (12) that revealed a metabolic acidosis and hypotension caused by renal loss of HCO K ϩ channels of the TASK subfamily are blocked by protons by titration of a histidine (N in TALK channels) residue in the first P domain (6-8), making them responsive to pH in the physiological range. The pH-sensing mechanism ...

ClC‐2 is a ubiquitously expressed, two‐pore homodimeric Cl− channel opened by hyperpolarisation. Little is known about its gating mechanisms. Crystallographic and functional studies in other ClC channels suggest that a conserved glutamate residue carboxylate side‐chain can close protopores by interacting with a Cl−‐binding site in the pore. Competition for this site is thought to provide the molecular basis for gating by extracellular Cl−. We now show that ClC‐2 gating depends upon intra‐ but not extracellular Cl− and that neutralisation of E217, the homologous pore glutamate, leads to loss of sensitivity to intracellular Cl− and voltage. Experiments testing for transient activation by extracellular protons demonstrate that E217 is not available for protonation in the closed channel state but becomes so after opening by hyperpolarisation. The results suggest that E217 is a hyperpolarisation‐dependent protopore gate in ClC‐2 and that access of intracellular Cl− to a site normally occupied by its side‐chain in the pore stabilises the open state. A remaining hyperpolarisation‐dependent gate might correspond to that closing both pores simultaneously in other ClC channels.