Molecular mechanisms of regulation of L-type Ca channels

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Activity of the cardiac L-type Ca^<2+> channel (Ca_v1.2) is modulated by dual feedback mechanisms, i.e., Ca^<2+>-dependent facilitation (CDF) and Ca^<2+>-dependent inactivation (CDI). Although calmodulin (CaM) has been suggested to mediate both CDF and CDI, Ca^<2+>/CaM-dependent protein kinase II (CaMKII) is also suggested to play a primary role in CDF. In this study, we investigated the roles and relations of Ca^<2+>, CaM and CaMKII in the basal activity, CDF and CDI using the patch-clamp method in guinea-pig ventricular myocytes. In the cell-attached mode, inhibitors of CaM significantly reduced both CDF and CDI, whereas those of CaMKII only modulated the time courses of CDF and CDI. In the inside-out mode, CaM (0.1-3μM) + ATP (2.4-3 mM)(at [Ca^<2+>]_i <10 nM) produced dose-dependent channel activity with a bell-shaped relationship between [CaM] and channel activity and with a maximum activation of 200-300% of control. Increasing of [Ca^<2+>]_i (~500 nM) shifted the [CaM]-channel activity curve toward left (i.e., to lower [CaM]). Thus, at a fixed concentration of CaM (e.g. 0.5 μM), Ca^<2+> showed biphasic effects: facilitation at [Ca^<2+>]_i <500 nM, and inactivation at [Ca^<2+>]_i >500 nM. This Ca^<2+>-dependent effect of CaM was considered to represent CDF and CDI. Based on these data, a simple model for Ca^<2+>-and CaM-dependent modulation of the Ca_v1.2 channel has been proposed, in which the channel had two CaM-binding sites, one for the basal activity and CDF and the other for CDI. In conclusion, it is suggested that CaM plays key roles in maintaining the basal activity and in producing CDF and CDI of the channel, and that CaMKII and Ca^<2+> modulate the interaction between the channel and CaM.