When the internal solution was alkalinized from pHi7.3 to 7.7 and 8.0, the channel activity increased to 120 and 200 %, respectively. play pivotal roles in determining Gambogic acid the resting membrane potential (Kir2.0), in G protein- or intracellular 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 (Bondet al.1994), though with Kir4.1 it may form a functional K+channel inXenopusoocytes (Pessiaet al.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 occurin vivo? We found that in addition to differences in the conductance and kinetic properties between the two channels, the heteromeric Kir4.1/Kir5.1 channel was much more sensitive to internal pH than the homomeric Kir4.1 channel. Using specific antibodies for Kir4.1 and Kir5.1 subunits, we also found that rat kidney and brain contained both Kir subunit proteins, but they were co-immunoprecipitated only from kidney. Therefore the heteromeric Kir4.1/Kir5.1 channel existsin vivo, at least in kidney, and may be involved in intracellular pH-dependent alteration of renal handling of ion transport. == METHODS == == Transient expression of Kir4.1 and Kir5.1 in HEK293T cells == Kir5.1 cDNA was cloned from a rat kidney cDNA library as described previously (Takumiet al.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 Gambogic acid 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 4896 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 1 kHz (-3 dB) through an eight-pole Bessel filter, sampled at 5 kHz, and analysed off-line on a Gambogic acid 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 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 366379 of Kir4.1 and 169187 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. Rabbit Polyclonal to Tip60 (phospho-Ser90) 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 regulations of the Animal Care Committee of Osaka University Medical.