In resting cardiomyocytes, the intracellular concentration of Ca2+ ([Ca2+]i) is 20,000 times less than its concentration in the extracellular environment (<0.1 m M vs 1-2 mM) and the cell interior is electronegative (-50 to-90 mV). Thus, there is an electrochemICal gradient that favors the entry of Ca2+ into the cell. When the cardiomyocytes is depolarized, the [Ca2+]i increases to 0.3-1 m M due to the entry of extracellular Ca2+ through L-type channels of the sarcolemma (and to a much lesser extent via the Na++-Ca2+ exchanger) and/or the release of Ca2+ from intracellular stores, mainly the sarcoplasmic reticulum (Ca2+ induced Ca2+ release).
The Ca2+ entry from the extracellular space through L-type channels in cardiac cells generates an inward current (INa+) that is required for excitation-contraction coupling in the mammalian myocardium, transmitter release and hormone secretion, excitation-transcription coupling and plays a key role in AP generation and in regulating automaticity in the sino-atrial (SA), the conduction velocity through the atrio-ventricular node, the plateau (phase 2) of the AP and certain forms of focal arrhythmogenic activity (abnormal automaticity developed in ischemic-depolarized cells and early afterdepolarizations generated in situations in which prolongs the QT interval of the ECG).
The cardiac L-type calcium channel is a hetero-oligomeric protein consisting of a pore-forming α1 sununit (α1C, 200 kDa), which is able to form functioNa+l channels, and a set of auxiliary or regulatory subunits: a disulfide-linked subunit dimer a 2/ d (175 kDa) and intracelular β1-3 (55-60 kDa) subunits (Figure) which are encoged by the genes CACNa+1C (or CACNa+1D), CACNa+2D1 (or CACNa+2D3) and CACNB1-3, respectively (Nerbonne y Kass, 2005; Bodi y cols., 2005) (Figure). β Subunits are tightly associated at the cytoplasmic face of α1 (through the I–II linker), whereas α2δ subunits are GPI-anchored to the plasma membrane and interact with extracellular domains of α1.
The α1C subunit (Cav1.2, 2179 amino acids) consists of four homologous domains (I-IV), each with six transmembrane segments (S1-S6), which associate in the cytoplasmatic membrane to form the ion-conducting pore. This subunit also contains the voltage sensor of the channel, , comprised by transmembrane segments S1–S4 in each homologous repeat (I–IV), the pore-forming region comprised by S5 and S6 segments together with their connecting linker, which contains helical regions contributing to the formation of the selectivity filter. A Ca2+-selective pore region is located between S5 and S6 (Caterall, 2000). The high (100-fold) selectivity of the channel for Ca2+ over small monovalent cations is related to a conserved ring of glutamates (EEEE) located in the pore that affinity Ca2+ (KD =500 nmol/L) forming the filter channel selectivity (Koch, et al., 2000). The interaction between a 1 and the cytoplasmic b subunit is mediated by a well-defined sequence motifs on b (BID, b subunits interaction domain) and on the I-II cytoplasmic linker of a 1 (AID). The possible interaction sites between the a subunit and the ryanodine receptor (RyR2) of the sarcoplasmic reticulum are localized in the linker between DII and DIII. . The α1C subunit also harbors the binding sites for channel-modulating drugs.
The long C-termiNa+l of the α1 subunits serves as an important modulatory domain and is a target of numerous protein–protein interactions. The C-terminus contains a modulatory domain that interferes with calmodulin (CaM) modulation. This C-termiNa+l modulator (CTM) consists of two putative α-helices: one on the C-termiNa+l end (termed DCRD) and one immediately after the main CaM interaction site (PCRD), the so-called IQ motif. The proximal C-terminus also contains an EF-hand and the binding sites for calmodulin kiNa+se II (CaMKII) and protein phosphatase 2A (Zuhlke, et al., 1999). Upon Ca2+ binding CaM undergoes a conformatioNa+l change that promotes iNa+ctivation (so-called Ca2+-dependent iNa+ctivation, CDI) by interaction with additioNa+l C-termiNa+l (Christel and Lee, 2012). Thus, CDI is an important autoinhibitory mechanism preventing excessive Ca2+ influx. Like the voltage-dependent iNa+ctivation (VDI) occurring during depolarization, CDI appears to involve conformatioNa+l rearrangements of the intracellular channel mouth.
The A-kiNa+se anchoring protein (AKAP15) inteacts with the C-terminus by a conserved leucine zipper-like motif and targets protein kiNa+sa A (PKA) near the PKA phosphorylation site (serine1928) (Hulme, 2003). Activation of b-adrenergic receptors greatly increases the L-type Ca2+current through Cav1.2 channels, which requires phosphorylation by cAMP-dependent protein kiNa+se (PKA) anchored via AKAP15. PKA activation phosphorylates Cav1.2 α1 residues serine-1700 and threonine-1704, which are located within the PCRD helix facing the interface with DCRD. Disruption of PKA anchoring to Cav1.2 channels markedly inhibits the beta-adrenergic regulation of Cav1.2 channels via the PKA pathway in ventricular myocytes. The distal C-terminus cleaves from the α1 subunit and forms a complex with the truncated α1C subunit through the proximal and distal C-termiNa+l regulatory domains (PCRD and DCRD) and serves as a potent autoinhibitory domain. Formation of the autoinhibitory complex greatly reduces the coupling efficiency of voltage sensing to channel opening and shifts the voltage dependence of activation to more positive potentials (Hulme, 2006)
The β subunits (55 kDa) are cytosolic proteins that assemble with α1-subunits and regulate the expression of functioNa+l cell surface L-type Four different β-subunit-encoding genes, CACNB1, CACNB2, CACNB3, and CACNB4, which encode the β1-4 subunits, respectively, have been identified. In general, coexpression of β subunits modulates the biophysical properties of the α1 subunits, producing a leftward shift of the current-voltage relationship, which is consistent with the involvement of the S4 region of the α1 subunit voltage-sensor region.
The β2 subunit seems to be the most prominently expressed in the heart (Perez-Reyes et al., 1992). Its interaction with the α1-subunit is mediated by a well-defined sequence motifs on the b subunit (BID, β subunits interaction domain) and a highly conserved sequence motif in α1-subunits (α subunit interaction domain or AID) located in the I-II cytoplasmic linker (Pragnell y cols. 1994). The coexpression of the β and α1 subunits lend to a 10-fold increase in ICa density by increasing cell surface channel expression and channel open probability, and/or by stabilizing the α1-β channel complexes in the cell membrane, accelerates channel activation and iNa+ctivation kinetics and participates in the modulation of the Ca2+ channel induced by β-adrenergic stimulation (Mikala et al., 1998, Yamaguchi et al., 1998; Wei et al., 2000). The β subunit also antagonizes an endoplasmic reticulum retention sigNa+l located in the I-II linker of the α1 subunits facilitating the intracellular trafficking of α1C subunits toward the plasma membrane and its insertion in the proper geometry (Bichet et al., 2000; Yamagichi et al., 1998).
The C termiNa+l contains a 153-aa sequence in the human cardiac β2 subunits that is essential for modulating Ca2+ channel function and interaction with the α1C subunits (Kovrisnki et al., 2005) and a region that is involved in PI3K (phosphatidylinositol 3-kiNa+se)-induced increases of Cav1.2 (rat brain) channel density. PI3K increases ICa density by promoting the translocation of GFP-tagged Cav channels to the plasma membrane an effect mediated by phosphatidylinositol 3,4,5-trisphosphate–activated protein kiNa+se B (Akt/PKB) and that specifically requires β2 subunits (Viard et al., 2004).
The α2δ subunit (143 kDa), encoded by CACNa+2D1,CACNa+2D2,CACNa+2D3, and CACNa+2D4 genes, is transcribed from a single gene, translated and proteolytICally processed into α2 (N-terminus) and δ (C-terminus, extracellular) subunits that remain linked through disulfide bonds. The α2 domain is located extracellularly, and the δ subunity has a single transmembrane region with a very short intracellular part. Coexpression of α2δ with α1C subunit causes a 2-fold increase in expression of dihydropyridine binding sites, gating currents and ICa (Nerbonne y Kass, 2005), shifts the voltage dependence of activation of α1-β encoded channels and accelerates current activation and iNa+ctivation (Bangalore et al., 1996; Gurnett et al., 1996; Singer et al., 1991). It is probable that the α2/δ and β subunits “drive” the α1C subunit to the membrane in the correct insertion mode.
L-type Ca2+ channels activate-open on depolarization to membrane potentialspotentials positive to approximately -40 mV and the current amplitude peaks at around 0 mV. The channels activated very rapidly during depolarization (reaching a peak withing 2-7 ms) and remained open during the plateau phase of the AP generating the ICa. Channels iNa+ctivate over a time course of several tens of milliseconds to seconds. Theoverlap between the activation and iNa+ctivation curves indicates a “window current”. When the cardiac AP are very long (prolonged QT interval), as the membrane potential enter in this region (between -15 and -40 mV) the ICa that was largely iNa+ctivated during the plateau phase can be partly reactivated as the [Ca2+]i declines. This reactivation can cause a net depolarization inducing early depolarizations and triggered focal activity.
The iNa+ctivation of the channels is time, voltage-and Ca2+-dependent (Kass y Sanguinetti, 1984; Lee y cols., 1985). The Ca2+-dependent iNa+ctivation is a physiologICal process that provides a counregulatory mechanism to avoid an excessive Ca2+ entry into the cardiomyocytes. Calmodulin seems to play a central role in this process. Calmodulin appears to be prebound to the C-termiNa+l of the a 1C subunit at rest, near the IQ motive and acts as the Ca2+ sensor. On depolarization, Ca2+ enters the cells and it binds to the high-affinity binding sites on the C-termiNa+l lobe of calmodulin, which relieves the EF-hand-mediated inhibition of the I-II linker (channel blocker particle), allowing its interaction with the pore and the iNa+ctivation of the channel (Kim et al., 2004).
The recovery from iNa+ctivation is also voltage-and Ca2+-dependent very fast when the membrane potential is almost completely repolarized and at a given membrane potential elevated [Ca2+]i levels slow the recovery from iNa+ctivation. Thus, the combiNa+tion of slow or incomplete decine in [Ca2+]i during the diastole and reduced diastolic intervals, as often occur in heart failure or AF, decreases ICa availability.
Mutations in the CACNa+1C gene encoding Cav1.2 are associated to LQT8 (Tymothy syndrome), BrS3, SQTS4 and ERS2, mutations in the CACNa+2D1 encoding the a2d subunit in the SQTS6 and in the ERS4, mutations in the CACNB2B gene encoding the b2 subunit in the SBr4, the SQTS5 and the ERS3.
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