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Studies of Ion Transport at Extreme Temperature
Author(s) -
Cuppoletti John,
Birn Sarah S,
Tewari Kirti P,
Chakrabarti Jayati,
Malinowska Danuta H
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1201.24
Subject(s) - hek 293 cells , transfection , ion channel , biophysics , membrane , hyperthermophile , chemistry , ion transporter , ion , membrane potential , biochemistry , biology , gene , receptor , organic chemistry , archaea
Biological transport proteins have potential for use in many biotechnology applications. However, high temperatures or harsh chemical environments could limit their use. This study examines the stability of ion channels at high temperature. Preliminary studies using gramicidin showed H + transport at unexpectedly high temperature. In the present study a ClC‐like channel from the archeobacter hyperthermophile, Methanococcus jannaschii (mj) which grows at a temperature optimum of 85°–90°C, was cloned from mj genomic DNA. It was ligated into pcDNA3.1/V5‐His TOPO and stably expressed in HEK293 cells. Cl − currents were studied by whole cell patch clamp electrophysiology. ClCmj‐expressing HEK293 cells (but not mock transfected cells) exhibited voltage activated Cl − currents which were inhibited by 500 μM CdCl 2 . When membranes were isolated from ClCmj‐expressing HEK293 cells and fused to planar lipid bilayers, single Cl − channels were evident at room temperature (25°C) which persisted at elevated temperatures. No channels were evident with membranes from mock transfected HEK293 cells. These studies demonstrate that ion channels from these organisms will be functional at elevated temperatures as required for applications in biotechnology. Supported by AFOSR.