ICln ion channel splice variants in Caenorhabditis elegans: voltage dependence and interaction with an operon partner protein.
ICln is an ion channel identified by expression cloning using a cDNA library from Madin-Darby canine kidney cells. In all organisms tested so far, only one transcript for the ICln protein could be identified. Here we show that two splice variants of the ICln ion channel can be found in Caenorhabditis elegans. Moreover, we show that these two splice variants of the ICln channel protein, which we termed IClnN1 and IClnN2, can be functionally reconstituted and tested in an artificial lipid bilayer. In these experiments, the IClnN1-induced currents showed no voltage-dependent inactivation, whereas the IClnN2-induced currents fully inactivated at positive potentials. The molecular entity responsible for the voltage-dependent inactivation of IClnN2 is a cluster of positively charged amino acids encoded by exon 2a, which is absent in IClnN1. Our experiments suggest a mechanism of channel inactivation that is similar to the "ball and chain" model proposed for the Shaker potassium channel, i.e. a cluster of positively charged amino acids hinders ion permeation through the channel by a molecular and voltage-dependent interaction at the inner vestibulum of the pore. This hypothesis is supported by the finding that synthetic peptides with the same amino acid sequence as the positive cluster can transform the IClnN1-induced current to the current observed after reconstitution of IClnN2. Furthermore, we show that the nematode ICln gene is embedded in an operon harboring two additional genes, which we termed Nx and Ny. Co-reconstitution of Nx and IClnN2 and functional analysis of the related currents revealed a functional interaction between the two proteins, as evidenced by the fact that the IClnN2-induced current in the presence of Nx was no longer voltage-sensitive. The experiments described indicate that the genome organization in nematodes allows an effective approach for the identification of functional partner proteins of ion channels.
Fürst
Johannes
J
Department of Physiology, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.
Ritter
Markus
M
Rudzki
Jakob
J
Danzl
Johann
J
Gschwentner
Martin
M
Scandella
Elke
E
Jakab
Martin
M
König
Matthias
M
Oehl
Bernhard
B
Lang
Florian
F
Deetjen
Peter
P
Paulmichl
Markus
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins; D002874:Chromosome Mapping; D017931:DNA Primers; D004285:Dogs; D015640:Ion Channel Gating; D007473:Ion Channels; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D012326:RNA Splicing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D029742:Caenorhabditis elegans Proteins; D017931:DNA Primers; D007473:Ion Channels; D011506:Proteins; C498730:ICLn protein, C elegans