Actividades Académicas









































Seminario interdisciplinario: Molecular screening of chloride channels in single interstitial cells of cajal

Día: 28-JAN-13 12.00. Auditorio del Edificio ALFA.

The interstitial cells of Cajal (ICCs) are responsible for the pacemaker activity in the gut. The motor patterns developing in the intestine are driven by rhythmic membrane potential changes in ICCs known as slow waves. The slow waves are very likely the product of coordinated action of ion channels, exchangers and transporters. To date, several conductances have been identified to play a role in the generation of slow waves, including high conductance chloride currents (HCCC) generated by a channel of unidentified molecular nature. Very recently it has been proposed that the HCCC could be actually a maxi channel, given its properties of multiple subconductances, activation by patch excision and flickering activity, however, the molecular nature of the maxi channel is unknown. Objective: Given the importance of the maxi channel to understand the function of ICC and the generation of the pacemaker activity, we proposed to perform a chloride channel profiling in these cells using the RT-PCR. The different ICC populations are accompanied by several other cell types like neurons, smooth muscle cells and more composing the different layers of the small intestine and colon , therefore a pure culture of ICC has been proven difficult to obtain. To solve this problem our objective was to perform single cell PCR. Methods: In this work we report the validation of a RT-PCR essay that enables the molecular profiling of the ICC transcripts at the single cell level. Based on a microarray set of data from enriched ICC populations and bioinformatics analysis, we decided to profile several chloride channels as maxi anion channel candidates in primary cultures of ICC. Immunohistochemistry was also performed in frozen section and whole mount preparations of small intestine. Results: Of all collected cells, we chose those expressing c-kit or Tmem16A, specific markers for ICC, to look for chloride channels. Here we report the presence of the Calcium Activated Chloride Channel (Clca1), Chloride Channel 2 (Clcn2) and Chloride Channel 3 (Clcn3) in single ICC in culture, whereas Chloride Channel 5 (Clcn5) was not detected. In addition we report the presence of the three porins Vdac1 and Vdac3 in cultured ICC as well as Vdac2 from in situ ICC. We did not find the plasmalemmal isoform of Vdac1 in cultured ICC. The immunolabeling shows discrete labeling of porins in ICC. In addition we report the presence of another member of the Tmem16 family, Tmem16F in cultured ICC. Conclusions: Several families of chloride channels remain to be tested by this technique. We encourage the continuation of gene expression profiling experiments to elucidate the channel diversity of ICC and propose Vdac porins as possible candidates for the maxi channel present in these cells.

Co-directores de Tesis: Dr. Carlos Barajas López, DBM-IPICYT / Dr. Jan Huizinga (Mc. Master University)
Comité Tutoral:
Dra. Irene Castaño Navarro, DBM-IPICYT
Dr. Juan Francisco Jiménez Bremont, DBM-IPICYT

Expositor :

Raúl Loera Valencia

Institución :

Estudiante de la DBM

Información relativa a la división de:
Biología Molecular