Acid-Induced Chloride Current in Distal Convoluted Tubule

Acid-Induced Chloride Current in Distal Convoluted Tubule

Tuesday, March 1, 2016 Invertebrate-specific gap junction proteins, termed innexins, form a large family of four-transmembrane proteins. These protein...

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Tuesday, March 1, 2016 Invertebrate-specific gap junction proteins, termed innexins, form a large family of four-transmembrane proteins. These proteins oligomerize to constitute intercellular channels that allow for the passage of small signaling molecules associated with neural and muscular electrical activity. In contrast to the large number of structural and functional studies of vertebrate connexin gap junction channels, few structural studies of recombinant innexin channels have been published. Here we show a three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. An N-terminal deletion INX-6 construct in which amino acids 2 through 19 were removed (INX-6-deltaN) was crystallized in lipid bilayers. The threedimensional reconstruction based on cryo-electron crystallography revealed that the two-dimensional crystals of INX-6-deltaN comprise two lipid bilayers, including fully docked gap junction channels. A single INX-6-deltaN gap junction channel comprises 16 subunits, a hexa-decamer, in contrast to vertebrate connexin channels, which comprise 12 subunits. Two bulb densities were observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. The former is reminiscent of the plug observed in the connexin26 mutant structure we previously reported. A fluorescent dye transfer assay revealed that INX-6-deltaN junction channels were essentially impermeable. These findings imply the structural diversity of gap junction channels among multicellular organisms, and provide insight into the functional properties characteristic of invertebrates. 1742-Plat Access of Metal Ions and Methanethiosulfonate Reagents to the CalciumGated Connexin Hemichannel Pore: Implications for the Location of the Gate William I. Lopez, Jaya Ramachandran, Andrew L. Harris, Jorge E. Contreras. Pharmacology, Physiology and Neuroscience, Rutgers Medical School, Newark, NJ, USA. How connexin hemichannels open and close the pore in response to changes in voltage and extracellular calcium concentrations is still unknown. Previous work has shown that conformational changes at the extracellular entrance of the pore are critical for hemichannel gating by calcium and voltage. Recently, we found that negatively charged residues in this region could interact with calcium ions to produce occlusion of the pore. These residues form a ring, raising the possibility that a calcium-bound gating ring at the entrance of the pore forms a gate. To test whether such a gating ring serves as a physical gate that prevents the access of ions and small metabolites, we assessed the calcium state-dependent accessibility of Cd2þ and MTSES to a substituted cysteine in Cx26 (Cx26G45C) located below the residues that form the ring. Extracellular application of MTSES to Xenopus oocytes expressing Cx26G45C mutants modified hemichannel currents, indicating accessibility to this residue. However, the estimated reaction rate was independent of the extracellular calcium concentration (i.e., the reaction rate was the same whether the channels were open or were closed by calcium). Similarly, Cd2þ accessibility to residue G45C was nearly independent of extracellular Ca2þ concentration. These results indicate that the calcium-gating ring does not prevent access of ions or metabolites to the inner pore and that the physical gate is located deeper into the pore from, on the intracellular side of the G45C residues and the calcium-gating ring. R01GM099490/R01GM101950. 1743-Plat Measuring Proton Depletion in the Vicinity of Proton Channels Leon D. Islas1, Victor De la Rosa-Jimenez2, Esteban Suarez1, Gisela Rangel1. 1 Physiology, School of Medicine, National Autonomous University of Mexico, Mexico City, Mexico, 2Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA. Proton channels have evolved to provide a pH regulatory mechanism, affording the extrusion of protons from the cytoplasm at all membrane potentials. Previous evidence has suggested that channel-mediated acid extrusion could significantly change the local concentration of protons in the vicinity of the channel. In this experiments we directly measure the proton depletion caused by activation of Hv1 proton channels, using patch-clamp fluorometry recordings from channels labeled with the Venus fluorescent protein at intracellular domains. The fluorescence of the Venus protein is very sensitive to pH, thus behaving as a genetically encoded sensor of local pH. Eliciting outward proton currents increases the fluorescence intensity of Venus. This dequenching is related to the magnitude of the current and not to channel gating and is dependent on the pH gradient. Increasing or decreasing the distance of Venus to the membrane or placing the fluorescent protein at the amino or carboxy terminus did no produce different fluorescence behavior, indicating that the depletion microdomain is ˚ , which is an estimate of the distance of the Venus protein larger than 90 A from the inner face of the membrane. Changing the pH-dependent properties

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of Venus through mutations allows tuning of the pH sensitivity of the Venus-Hv1 construct. Our results provide direct evidence of local proton depletion due to flux through the proton-selective channel. 1744-Plat Acid-Induced Chloride Current in Distal Convoluted Tubule William C. Valinsky1, Rhian M. Touyz2, Alvin Shrier1. 1 Physiology, McGill University, Montreal, QC, Canada, 2Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom. The distal convoluted tubule (DCT) of the kidney is responsible for the finetuning of urinary filtrate before excretion. The DCT is known to facilitate the movement of Na, K, Ca, Mg, and Cl via transporters and ion channels. Recent research has focussed on the functional activity of TRP channels in the DCT, with heavy emphasis on TRPM6/7 (Mg at neutral pH 7.4; Na at acidic pH) and TRPV5/6 (Ca). Several studies have suggested that these channels should give rise to large macroscopic currents in DCT cells. Therefore we undertook this study to determine the ionic species representing the dominant current in mouse DCT (MDCT) cells via whole-cell voltage-clamp electrophysiology. Using ionic permeation studies and analysis of reversal potential changes, our results unequivocally demonstrate that MDCT cells exhibit a significant Cl current under both neutral and acidic extracellular solutions. We further identified that this current is rapidly activating, differentiating it from the endogenous acid-induced anion current discovered in HEK293 cells. We noted that currents elicited under acidic pH were substantially larger than those at neutral pH, and thus hypothesized that extracellular acid induced a different, larger Cl current in MDCT cells. This was supported pharmacologically, where only the acidinduced Cl current was blocked by furosemide, a clinically prescribed loopdiuretic. To further understand the significance of a large acid-induced Cl current in the late nephron, we investigated inner medullary collecting duct (IMCD) cells as they represent the most acidic portion of the nephron. IMCD current was not only acid-induced, but shared an identical I-V relationship, pH sensitivity, and time-course of activation with the acid-induced induced Cl current discovered in MDCT cells. We thus conclude that the late nephron contains a large acidinduced Cl current that may play a significant role in anion reabsorption. 1745-Plat Modulation of the Calcium-Dependent Chloride Channel TMEM16A by Extracellular Protons Silvia Cruz-Rangel1, Jose´ J. De Jesu´s-Pe´rez1, Criss Hartzell2, Patricia Pe´rez-Cornejo3, Jorge Arreola1. 1 Institute of Physics, Univ. Autonoma de San Luis Potosi, San Luis Potosi, Mexico, 2Cell Biology, Emory University School of Medicine, Atlanta, GA, USA, 3Physiology, Universidad Auto´noma de San Luis Potosı´ School of Medicine, San Luis Potosi, Mexico. TMEM16A, the pore forming subunit of the Ca2þ-dependent Cl- channel (CaCCs) plays an important role in physiological processes such as fluid secretion, muscular contractility and thermal nociception. This channel is activated in response to increments in intracellular [Ca2þ] and membrane depolarization. Previous studies established that endogenous CaCCs are regulated by extracellular protons ([Hþ]o). In this work the effect of [Hþ]o on the TMEM16A activity was evaluated using the whole-cell patch clamp technique. Stably transfected HEK293 cells were dialyzed with a solution containing 140 mM Cl- and 0.2 mM Ca2þ. We found that [Hþ]o regulates the chloride current (ICl) in a bimodal manner. Decreasing [Hþ]o from 107.3 to 1010 M decreased ICl without changing the activation kinetics. In contrast, increasing the [Hþ]o from 107.3 to 106 M increased ICl; but a further increase of [Hþ]o to 105, 104.5 or 104 M reduced ICl to 37% respect to 107.3 M. The effect of protons on ICl peaked at 105.5 M, were independent of the conformational state (closed or open) of the channel as well as of membrane voltage. The bimodal regulation suggests the existence of two titrable sites with pK values of 7.1 and 5.2, respectively. To verify this we generated the following mutants E362Q, E368Q, E623Q, E624Q, H402Y, H802Y, and H807Y, but none of the mutations prevented the effects of [Hþ]o. Then to investigate if the effect of [Hþ]o depended on the degree of TMEM16A activation we dialyzed cells with different [Ca2þ]i. Surprisingly we found that increasing the [Ca2þ]i produces a strong reduction of the Hþ effect, suggesting that extracellular protons regulate TMEM16A by altering the open probability of the channel. Supported by grant 219949 from CONACyT. 1746-Plat Unusual Ion Pathway Architecture of the Dual-Topology Fluoride Channel Fluc Nicholas B. Last, Christopher Miller. Department of Biochemistry, Brandeis Univ., Waltham, MA, USA. Fluoride is an ubiquitous environmental ion. Under mildly acidic conditions, the weakly acidic character of hydrofluoric acid allows it to pass through the