Cloning and characterization of a novel ion channel

Calcium influx through voltage-activated Ca{dollar}sp{lcub}2+{rcub}{dollar} channels (VACCs) plays essential roles in various physiological functions such as: membrane depolarization, muscle contraction, hormone secretion, synaptic transmission, cell viability, and gene expression. Molecular cloning and expression studies of Ca{dollar}sp{lcub}2+{rcub}{dollar} channel {dollar}alpha1{dollar} subunits have uncovered new aspects about structure, diversity, pharmacology, and biophysical properties of Ca{dollar}sp{lcub}2+{rcub}{dollar} channels. However, there still remain Ca{dollar}sp{lcub}2+{rcub}{dollar} channels that have not been cloned yet.; We searched for a Ca{dollar}sp{lcub}2+{rcub}{dollar} channel-like sequence from the Genbank using the BLAST (Basic Local Alignment Search Tool) program. A candidate for a novel Ca{dollar}sp{lcub}2+{rcub}{dollar} channel was found in a Caenorhabditis elegans cosmid (C27f2). Reverse transcription-polymerase chain reaction (RT-PCR) cloning was applied to clone a mammalian version of this putative channel. PCR primers derived from the C. elegans sequence amplified a PCR product (Rb21) whose deduced sequence was similar to the C. elegans sequence. The full-length cDNA of Rb21 was obtained by screening a rat brain cDNA library. The cloned full-length cDNA contains 6820 bases. The open reading frame is 5,214 bases and the molecular weight of the deduced amino acid sequence is about 205 kdaltons.; The deduced amino acid sequence of the Rb21-channel is homologous to reported Ca{dollar}sp{lcub}2+{rcub}{dollar} and Na{dollar}sp+{dollar} channels, but cannot be classified into any type of established ion channels. Structural analyses including hydropathy profile and matrix homology plots suggested that the Rb21-channel has four repeated domains, each of which contains six putative membrane spanning segments and a pore loop. According to a phylogenetic tree, the Rb21-channel might be an ancestral precursor of Ca{dollar}sp{lcub}2+{rcub}{dollar} and Na{dollar}sp+{dollar} channels. Northern blot analyses showed that the size of detected transcripts is about 6.9 kb and Rb21-channel transcripts are expressed predominantly in the brain, moderately in the heart, and weakly in the pancreas. Despite extensive expression studies, the expression of the Rb21-channel has not been detected as currents in Xenopus oocytes and HEK-293 cells.; Based on sequence and structural characteristics, I propose that the Rb21-channel might be a cation channel with unique voltage gating properties such as: a low voltage-activated Ca{dollar}sp{lcub}2+{rcub}{dollar} channel, a non-inactivating Na{dollar}sp+{dollar} channel, or a hyperpolarization-activated cation channel.