Abstract
The heart muscle responds to physiological needs with a short-term modulation of cardiac contractility. This process is determined mainly by properties of the cardiac L-type Ca(2+) channel (Ca(v)1.2), including facilitation and Ca(2+)-dependent inactivation (CDI). Both facilitation and CDI involve the interaction of calmodulin with the IQ motif of the Ca(v)1.2 channel, especially with Ile-1624. To verify this hypothesis, we created a mouse line in which Ile-1624 was mutated to Glu (Ca(v)1.2(I1624E) mice). Homozygous Ca(v)1.2(I1624E) mice were not viable. Therefore, we inactivated the floxed Ca(v)1.2 gene of heterozygous Ca(v)1.2(I1624E) mice by the α-myosin heavy chain-MerCreMer system. The resulting I/E mice were studied at day 10 after treatment with tamoxifen. Electrophysiological recordings in ventricular cardiomyocytes revealed a reduced Ca(v)1.2 current (I(Ca)) density in I/E mice. Steady-state inactivation and recovery from inactivation were modified in I/E versus control mice. In addition, voltage-dependent facilitation was almost abolished in I/E mice. The time course of I(Ca) inactivation in I/E mice was not influenced by the use of Ba(2+) as a charge carrier. Using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid as a chelating agent for intracellular Ca(2+), inactivation of I(Ca) was slowed down in control but not I/E mice. The results show that the I/E mutation abolishes Ca(2+)/calmodulin-dependent regulation of Ca(v)1.2. The Ca(v)1.2(I1624E) mutation transforms the channel to a phenotype mimicking CDI.
Highlights
26702 JOURNAL OF BIOLOGICAL CHEMISTRY types of regulation involve, as a key step, direct binding of the Ca2ϩ sensor protein calmodulin (CaM) to the Cav1.2 channel (4 –7), alongside with the action of CaM on Ca2ϩ/CaM-dependent protein kinase II (CaMKII) (2, 6, 8 –11)
Slow components of inactivation were not different in CMs from Ctr and I/E mice. These results suggest that the mutation of Ile to Glu at position 1624 of the Cav1.2 channel abolishes the effects of Ca2ϩ on inactivation of ICa, most likely because the channel has already been transformed to a phenotype mimicking Ca2؉-dependent inactivation (CDI)
We have shown that exchange of Ile with Glu in the CaM-binding motif (IQ) of the Cav1.2 channel gene is lethal to mice
Summary
26702 JOURNAL OF BIOLOGICAL CHEMISTRY types of regulation involve, as a key step, direct binding of the Ca2ϩ sensor protein calmodulin (CaM) to the Cav1.2 channel (4 –7), alongside with the action of CaM on Ca2ϩ/CaM-dependent protein kinase II (CaMKII) (2, 6, 8 –11). The I/E mutation abrogates CDF and CDI of L-type Ca2ϩ currents expressed in Xenopus oocytes [11]. It is unclear if the binding of CaM to the Cav1.2 channel is relevant for the function of the cardiac calcium channel in the behaving mouse. The results show that this mutation is lethal To overcome this problem, we generated mice with a conditional heart-specific I/E mutation in the Cav1.2 channel gene. Electrophysiological analysis of Cav1.2 channel currents in cardiomyocytes (CMs) from I/E mice revealed that the I/E mutation blocks CaM/CaMKII-mediated regulation of the Cav1.2 channel in the heart and induces a channel phenotype with permanent CDI characteristics
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