An interesting feature of the L-type Ca channels is facilitation which is characterized by an increase in current flow through these channels following strong and/or repetitive depolarizations. The long-term objective of this project is to understand the molecular mechanisms of voltage-dependent gating of the cardiac Ca channel as well as any changes that occur in this gating under normal and pathophysiologic conditions. The present project focuses on the initial opening or activation of these channels using patch clamp techniques and site-directed mutagenesis and chimera construction to study cloned Ca channel subunits expressed in HEK293 cells. Three hypotheses will be tested: 1) Voltage-dependent facilitation of heterologously expressed cardiac a1C Ca channels requires b subunit co-expression. 2) Specific and identifiable functional domains of the b subunit are essential for voltage-dependent facilitation. 3) A distinct component of intramembrane charge movement, Qf, underlies voltage-dependent facilitation.
Five specific aims will be examined: 1) To define voltage-dependent activation and facilitation of heterologously expressed a1C+a2-d Ca channels in the absence and presence of different b subunit isoforms. 2) To investigate the role of second messenger pathways in voltage-dependent facilitation. 3) To define the structural region(s) of the Ca channel b subunit necessary for voltage-dependent facilitation. 4) To characterize voltage-dependent intramembrane charge movement associated with a1C+a2-d+b Ca channels to identify a component of charge movement, Qf, responsible for facilitation. 5) To quantitate the difference in the number of elementary charges per channel comparing a1C+a2-d vs. a1C+a2-d+b channels in order to detect Qf.
| Kamp, T J; Hu, H; Marban, E (2000) Voltage-dependent facilitation of cardiac L-type Ca channels expressed in HEK-293 cells requires beta-subunit. Am J Physiol Heart Circ Physiol 278:H126-36 |
