Abstract
TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca2+-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca2+-induced measured macroscopic Cl− currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl− channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca2+-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.
Highlights
From the ‡Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany; §Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle, Magdeburger Strae 6, D-06097 Halle/Saale, Germany
The mTMEM16A(a) proteins were affinity-purified on a Ni-affinity resin, eluted in nondenaturing buffer, and analyzed using nonreducing SDS-PAGE, blue native polyacrylamide gel electrophoresis (BN-PAGE), and chemical cross-linking to determine their oligomeric state
N- or C-terminally His-tagged mTMEM16A(a) and a Tandem mTMEM16A(a) Homodimer are Functional in X. laevis Oocytes—To examine whether an N- or C-terminally positioned His-tag affects the function of mTMEM16A(a), Two-Electrode Voltage-Clamp (TEVC) measurements were performed
Summary
CaCC, Ca2ϩ-activated chloride channel; BN-PAGE, blue native PAGE; CLC, voltage-gated chloride channel; Cy5 NHS ester, cyanine 5 N-hydroxysulfosuccinimide ester; Endo H, endoglycosidase H; GlyR, glycine-activated receptor; 5HT3R, mouse serotonin type 3 receptor; Ni-NTA, nickel-nitrilo acetic acid; ORi, frog concentrations, were first identified in Xenopus laevis oocytes [1, 2], where they generate the fertilization potential that provides a fast electrical block to prevent polyspermy [3]. CaCC-activating stimuli include Ca2ϩ influx from the extracellular space through voltage-gated Ca2ϩ channels opened by oocyte depolarization to potentials around 0 mV [1], Ca2ϩ released from intracellular IP3-sensitive Ca2ϩ stores [8], or intracellular Ca2ϩ injection [5, 9]. The TMEM16 family includes ten members (designated A through K) that share a common topology, including long intracellular N- and C-terminal tails and eight membrane-spanning segments (designated M1 to M8) that are connected by intervening loops (Fig. 1A) Because of their role in anion transport and their eight-transmembrane, helical topology, TMEM16 proteins are designated as anoctamins [15]. The removal of exons 6b or 13 results in an approximately fourfold increase in Ca2ϩ sensitivity or a change in the time-dependent CaCC current activation at positive membrane potentials, respectively [16]. Partial results of this work were presented in a poster at the 24th Annual Symposium of The Protein Society [36]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
