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Protein and gene expression of Ca2+ channel isoforms in murine colon: effect of inflammation
Minho Kang1, Nemat Morsy, Xiaochun Jin
1Department of Physiology, University of Oklahoma, Health Sciences Center, Oklahoma City, OK 73104, USA.
Abstract:
L-Type voltage-dependent Ca2+ channels (L-VDCC) mediate calcium influx in response to membrane depolarization and regulate intracellular processes such as contraction, secretion, neurotransmission, and gene expression. Colonic inflammation significantly attenuates calcium currents in smooth muscle; however, the basis for this remains unclear. In this study we examined the protein and mRNA expression of two isoforms of Ca(v)1.2, encoded by either exon la or 1b. Both isoforms were detected by Western blots, immunohistochemistry and RT-PCR in smooth muscle cells. Neither the protein nor mRNA expression measured by real-time PCR of either isoforms was affected in colonic myocytes from dextran sulfate sodium-treated mice. In whole-cell voltage-clamp experiments, the amplitude of the calcium currents were decreased by almost 70% by inflammation. The calcium channel currents were attenuated by 50 +/- 3% by the c-src kinase specific inhibitor, PP2, in control cells but only 19 +/- 7% in cells from inflamed mice. These studies suggest that decreased calcium channel currents following colonic inflammation are not due to decreased expression but may result from altered regulation by the non-receptor cellular tyrosine kinase, c-src kinase.
Insights
Colonic inflammation reduces calcium channel currents in smooth muscle, not by decreasing expression, but through altered regulation by c-src kinase, impacting cellular calcium influx.
Area of Science:
- Physiology
- Molecular Biology
- Gastroenterology
Background:
- L-Type voltage-dependent Ca2+ channels (L-VDCC) are crucial for smooth muscle function.
- Colonic inflammation is known to reduce calcium currents, but the underlying mechanisms are not fully understood.
Purpose of the Study:
- To investigate the expression of Ca(v)1.2 isoforms (exon 1a and 1b) in colonic smooth muscle.
- To determine if inflammation affects Ca(v)1.2 expression or regulation.
Main Methods:
- Western blot, immunohistochemistry, and RT-PCR were used to assess Ca(v)1.2 protein and mRNA expression.
- Whole-cell voltage-clamp electrophysiology was employed to measure calcium currents.
- The effect of c-src kinase inhibition (PP2) on calcium currents was evaluated in control and inflamed conditions.
Main Results:
- Both Ca(v)1.2 isoforms (exon 1a and 1b) were expressed in colonic smooth muscle cells at both protein and mRNA levels.
- Colonic inflammation did not alter the expression of Ca(v)1.2 isoforms.
- Inflammation significantly decreased calcium channel current amplitude by approximately 70%.
- c-src kinase inhibition attenuated currents more in control cells than in inflamed cells, suggesting altered kinase regulation.
Conclusions:
- Decreased calcium channel currents in colonic inflammation are not caused by reduced Ca(v)1.2 expression.
- Altered regulation of L-VDCC by c-src kinase likely contributes to the diminished calcium currents observed during colonic inflammation.
