The Role of Region IVS5 of the Human Cardiac Calcium Channel in Establishing Inactivated Channel Conformation

Abstract

The role of inactivated channel conformation and use dependence for diltiazem, a specific benzothiazepine calcium channel inhibitor, was studied in chimeric constructs and point mutants created in the IVS5 transmembrane segment of the L-type cardiac calcium channel. All mutations, chimeric or point mutations, were restricted to IVS5, while the YAI-containing segment in IVS6, i.e. the primary interaction site with benzothiazepines, remained intact. Slowed inactivation rate and incomplete steady state inactivation, a behavior of some mutants, were accompanied by a reduced or by a complete loss of use-dependent block by diltiazem. L-type Ca 2ϩ channels of cardiac, skeletal, and smooth muscle play a central role in excitation-contraction coupling. It is also believed that these channels may participate in the pathophysiology of some cardiac arrhythmias, hypertension, and angina pectoris (1-4). Increases in [Ca 2ϩ ] i have been linked to a variety of changes in membrane properties that contribute to the changes in excitability, conduction, refractoriness, automaticity, and vascular resistance (1-4). In terms of excitation-contraction coupling, Ca 2ϩ enters the cell during depolarization through voltage-activated calcium channels and triggers contraction but also is responsible for activation of other cellular functions. In the course of sustained depolarization, Ca 2ϩ currents progressively undergo voltage-dependent inactivation with specific kinetics, primarily regulated by the membrane potential.Ca 2ϩ channels are the target for three main classes of drugs that include dihydropyridines (DHP), 1 phenylalkylamines (PAA), and benzothiazepines (BTZ). These molecules bind specifically to different sites of the Ca 2ϩ channel ␣ 1 subunit, and their binding domains are allosterically linked to each other (5-7). Ca 2ϩ channel antagonists, particularly diltiazem and verapamil, block calcium channels in a voltage-and use-dependent manner; thus the binding of the drug facilitates protein conformational changes that alter channel function by interfering with channel gating. These drugs serve as useful tools in dissecting molecular mechanisms of channel inactivation. Several amino acid residues that are involved in Ca 2ϩ antagonist binding, when mutated, change the electrophysiological properties of the channel, such as voltage-dependent inactivation. Single amino acids in segments IIIS6 and IVS6 (5, 8 -11) have been identified as determinants of inactivation. Mutation of key amino acids in these segments alters not only the high affinity drug-binding sites but also the complexity of the kinetic behavior of the channels, consequently changing use-dependent block particularly for the PAAs and BTZs (for review, see Ref. 12).Our previous finding showed that a segment in IVS5 of the human ␣ 1C (Ca v 1.2) subunit is critically involved in inactivation of the channel (13). Consequently, mutants constructed in this region lost the characteristic use-dependent block by PAA and BTZ and recovered from inactivation significantly faster a...

Molecular Determinants of Ca2+ Potentiation of Diltiazem Block and Ca2+-Dependent Inactivation in the Pore Region of Cav1.2

“…4B), suggesting that the EIVQ mutation did not disrupt the ability of diltiazem to bind to the inactivated state of the channel (Li et al, 1999). This characteristic of the EIVQ mutation contrasts with mutations in IVS5 of Ca v 1.2 that disrupted frequency-dependent block but also markedly disrupted voltage-dependent inactivation (Motoike et al, 1999;Bodi et al, 2002). Thus, neither the small changes in the voltage dependence of activation and inactivation nor changes in the ability of diltiazem to bind the inactivated state of EIVQ likely explain the marked loss of frequency-dependent block accumulation in EIVQ.…”

Roscovitine, a cyclin‐dependent kinase inhibitor, affects several gating mechanisms to inhibit cardiac L‐type (Ca(V)1.2) calcium channels

“…This was expected since the slowed recovery from inactivation ( τ Recov ∼72 ms) would not impact inhibition until the interval between stimuli was 100 ms. Thus, use‐dependent inhibition is not observed over the frequency range used to observe use‐dependent block of Ca (V) 1.2 current by phenylalkylamines and benzothiazepines (Hering et al , 1996; Johnson et al , 1996; Motoike et al , 1999; Bodi et al , 2002).…”

Negatively Charged Residues in the N-terminal of the AID Helix Confer Slow Voltage Dependent Inactivation Gating to CaV1.2

“…The observation that the voltage dependence of inactivation of E462R was affected by the host channel could stem from the intrinsic voltage-dependent properties in Ca V 1 versus Ca V 2 channels. Mutations in IVS5, IIS6, IIIS6, and IVS6 were reported to decrease VDI kinetics of Ca V 1.2 without any significant change in its voltage dependence of inactivation (Bodi et al, 2002;Shi and Soldatov, 2002). In one case, the acceleration of VDI kinetics brought by the F823A mutation in the IIS6 region of the rat Ca V 1.2 was accompanied by the hyperpolarization of both the voltage dependence of activation and the voltage dependence of inactivation (Stotz and Zamponi, 2001).…”