Related Experiment Videos
Identification of an mRNA species which encodes a voltage-operated Ca2+ channel in rat liver mRNA
B P Hughes1, K Both, L Harland
1Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, South Australia.
Abstract:
cDNA which encodes part of the alpha 1-subunit of the rabbit skeletal muscle L-type voltage-operated Ca2+ channel (VOCC cDNA) was employed to search for the presence in whole liver and hepatocytes of poly (A+) RNA homologous to mRNA which encodes VOCCs. Such homologous mRNA would be a candidate for mRNA which encodes the putative hepatocyte receptor-activated Ca2+ inflow system (RACIS). Northern blot analysis showed that poly (A+) RNA prepared from intact liver tissue, but not hepatocytes, contained a poly (A+) RNA species comparable in size to that which encodes the alpha 1-subunit of the L-type VOCC. It is concluded (a) that hepatocytes do not possess VOCCs or that the levels of VOCC poly(A+) RNA in hepatocytes are too low to be detected by Northern analysis and (b) that another cell type present in liver tissue does possess a VOCC. In a low stringency screen of a rat liver cDNA library employing VOCC cDNA as a probe, seven positive cDNA clones were obtained. While regions of the 2.3 kb cDNA insert from one of these clones showed sequence similarities with regions of VOCC cDNA, the 2.3 kb sequence did not appear to encode a Ca2+ channel. The present results suggest that the approach of low stringency cDNA library screening is unlikely to allow isolation of RACIS cDNA.
Insights
Hepatocytes lack detectable mRNA for voltage-operated calcium channels (VOCCs), suggesting another liver cell type expresses them. Current methods may not be suitable for isolating the receptor-activated calcium inflow system (RACIS) mRNA.
Area of Science:
- Molecular Biology
- Cell Physiology
- Biochemistry
Background:
- Voltage-operated calcium channels (VOCCs) are crucial for cellular calcium regulation.
- The receptor-activated calcium inflow system (RACIS) in hepatocytes is not fully characterized.
- Understanding calcium channel expression in liver cells is important for hepatic function.
Purpose of the Study:
- To investigate the presence of mRNA homologous to VOCCs in rabbit skeletal muscle and liver cells.
- To determine if hepatocytes express mRNA encoding VOCCs, a potential candidate for RACIS.
- To explore cDNA library screening for the isolation of RACIS cDNA.
Main Methods:
- Northern blot analysis of poly (A+) RNA from whole liver and isolated hepatocytes.
- Low stringency screening of a rat liver cDNA library using VOCC cDNA as a probe.
Main Results:
- Poly (A+) RNA homologous to L-type VOCC alpha 1-subunit mRNA was detected in whole liver but not in hepatocytes.
- A 2.3 kb cDNA clone from a rat liver library showed partial sequence similarity to VOCC cDNA but did not encode a calcium channel.
- Seven positive cDNA clones were identified in the low stringency screen.
Conclusions:
- Hepatocytes do not appear to possess detectable levels of VOCC poly (A+) RNA, or they are present at very low levels.
- A non-hepatocyte cell type within the liver tissue expresses VOCCs.
- Low stringency cDNA library screening may not be an effective strategy for isolating RACIS cDNA.