the single channel current-voltage relationship at different pHi levels

the single channel current-voltage relationship at different pHi levels. metabolism-dependent regulation of cell excitability (Kir3.0 and Kir6.0, respectively), and in transporting K+ ions in epithelial tissues and glial cells (Kir1.1 and Kir4.0). Kir5.1 does not belong to any of these subfamilies and its physiological roles are unknown (Bond 1994), though with Kir4.1 it may form a functional K+ channel in oocytes (Pessia 1996). To clarify the physiological role of Kir5.1, we addressed two questions: (1) what is the functional difference between homomeric Kir4.1 channels and heteromeric Kir4.1/Kir5.1 channels? and (2) does the heteromeric assembly of Kir5.1 and Kir4.1 occur 1995). The coding regions of rat Kir1.1, Kir4.1 and Kir5.1 cDNAs were subcloned into an expression vector, pCDNA3 (Invitrogen, San Diego, CA, USA). HEK293T cells were then transfected with the plasmid vectors using LipofectAMINE (Life Technologies, Gaithersburg, MD, USA). To express the Kir4.1/Kir5.1 heteromer, Kir4.1 and Kir5.1 cDNAs were co-transfected, with the Kir5.1 cDNA:Kir4.1 cDNA ratio being 5:1. Electrophysiological measurements were conducted 48C96 h after transfection. Electrophysiological recordings The currents flowing through the channels expressed in HEK293T cells were measured using the patch-clamp method in the whole-cell, cell-attached patch and inside-out patch configurations. Currents were measured using a patch-clamp amplifier (EPC-7, List Electronics, Darmstadt, Germany) and recorded on videocassette tapes with PCM converter system (RP-880, NF Electronic Circuit Design, Yokohama, Japan). The data were reproduced, low-pass filtered at Abiraterone (CB-7598) 1 kHz (-3 dB) through an eight-pole Bessel filter, sampled at 5 kHz, and analysed off-line on a computer. All experiments were performed at room temperature (22-24C). The pipette and bath solutions contained (mM): 90 KCl, 5 EGTA, and 50 Hepes potassium salt (pH 7.4). The bath solution for whole-cell recording contained (mM): 120 NaCl, 20 KCl, 5 EGTA, 2 MgCl2, and 5 Hepes potassium salt (pH 7.4). To prepare external or internal solutions with different pH values, Mes (< pH Abiraterone (CB-7598) 7), Hepes (pH 7-pH 8) and Tris buffers (> pH 8) were used. The pH of the solutions was adjusted to the desired value by adding NaOH for external solutions or KOH for internal solutions. Antibodies Polyclonal anti-Kir4.1 and anti-Kir5.1 antibodies were raised in rabbits against the synthetic peptides EKEGSALSVRISNV and LAKMATARKRAQTIRFSYF which correspond to amino acids 366C379 of Kir4.1 and 169C187 of Kir5.1, respectively. These antibodies were purified with antigenic peptide-coupled Sulfonlink resin (Pierce, Rockford, IL, USA) and specifically detected, using Western blotting analysis, Kir4.1 or Kir5.1 heterologously expressed in HEK293T cells. Both immunoreactivities were prevented by their respective antigenic peptides. Rabbit polyclonal anti-green fluorescent protein (GFP) and mouse monoclonal anti-FLAG M2 antibodies were purchased from Clontech Laboratories (Palo Alto, CA, USA) and Eastman Kodak (New Haven, CT, USA), respectively. Immunoblot analysis and immunoprecipitation analysis An adult male Spraque-Dawley rat was anaesthetized with ether and killed by decapitation, according to the Abiraterone (CB-7598) regulations of the Animal Care Committee of Osaka University Medical School. Membrane preparations of Kir4.1- and/or Kir5.1-transfected HEK293T cells and of several rat tissues were obtained. The membrane proteins were solubilized in a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 g ml?1 aprotinin, 100 g ml?1 PMSF, 0.02 % sodium azide, 0.1 % SDS, 0.5 % sodium deoxycholate, and 1 % Triton X-100. About Abiraterone (CB-7598) 40 g of the membrane proteins were separated using SDS-PAGE (10 %10 %) and transferred to PVDF (polyvinylidene di-fluoride) membranes. The membranes were incubated for 12 h at 4C with a preventing buffer filled with 80 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 % (w/v) skimmed milk and 0.2 % Triton X-100. These were after that incubated in preventing buffer filled with antibodies at a focus of 0.5 g ml?1 for 12 h in 4C. After incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (EnVision Plus; Dako, Carpinteria, CA, USA) diluted to at least one 1:500 (v/v) in preventing buffer, membranes had been washed once using a cleaning buffer (2 % Lubrol-PX in PBS) and double with PBS. Immunoreactive rings had been detected using a SuperSignal chemiluminescence immunostaining package (Pierce, Rockford, IL, USA). For immunoprecititation evaluation, proteins A-Sepharose beads had been pretreated with 1 % (w/v) BSA in the lysis buffer for 2 h at 4C. The proteins A-Sepharose beads had been after that incubated with antibodies (1 g antibodies per 20 l proteins A-Sepharose) in the lysis buffer for 4 h at 4C. The solubilized membrane fractions of every tissue, ready as defined above, had been incubated using the antibody-pretreated proteins A-Sepharose PRSS10 beads for 16 h at 4C. Pellets of proteins A-Sepharose beads were washed five situations using the lysis then.