Vladimir Yarov-Yarovoy

Cardioprotective Effect of Diazoxide and Its Interaction With Mitochondrial ATP-Sensitive K+ Channels Possible Mechanism of Cardioprotection

Previous studies showed a poor correlation between sarcolemmal K+ currents and cardioprotection for ATP-sensitive K+ channel (KATP) openers. Diazoxide is a weak cardiac sarcolemmal KATP opener, but it is a potent opener of mitochondrial KATP, making it a useful tool for determining the importance of this mitochondrial site. In reconstituted bovine heart KATP, diazoxide opened mitochondrial KATP with a K1/2 of 0.8 mumol/L while being 1000-fold less potent at opening sarcolemmal KATP. To compare cardioprotective potency, diazoxide or cromakalim was given to isolated rat hearts subjected to 25 minutes of global ischemia and 30 minutes of reperfusion. Diazoxide and cromakalim increased the time to onset of contracture with a similar potency (EC25, 11.0 and 8.8 mumol/L, respectively) and improved postischemic functional recovery in a glibenclamide (glyburide)-reversible manner. In addition, sodium 5-hydroxydecanoic acid completely abolished the protective effect of diazoxide. While-myocyte studies showed that diazoxide was significantly less potent than cromakalim in increasing sarcolemmal K+ currents. Diazoxide shortened ischemic action potential duration significantly less than cromakalim at equicardioprotective concentrations. We also determined the effects of cromakalim and diazoxide on reconstituted rat mitochondrial cardiac KATP activity. Cromakalim and diazoxide were both potent activators of K+ flux in this preparation (K1/2 values, 1.1 +/- 0.1 and 0.49 +/- 0.05 mumol/L, respectively). Both glibenclamide and sodium 5-hydroxydecanoic acid inhibited K+ flux through the diazoxide-opened mitochondrial KATP. The profile of activity of diazoxide (and perhaps KATP openers in general) suggests that they protect ischemic hearts in a manner that is consistent with an interaction with mitochondrial KATP.

The mitochondrial KATP channel as a receptor for potassium channel openers

The biochemical properties of the mitochondrial K channel are very similar to those of plasma membrane K channels, including inhibition by low concentrations of ATP and glyburide (Paucek, P., Mironova, G., Mahdi, F., Beavis, A. D., Woldegiorgis, G., and Garlid, K. D.(1992) J. Biol. Chem. 267, 26062-26069). Plasma membrane K channels are highly sensitive to the family of drugs known as K channel openers, raising the question whether mitochondrial K channels are similarly sensitive to these agents. We addressed this question by measuring K flux in intact rat liver mitochondria and in liposomes containing K channels purified from rat liver and beef heart mitochondria. K channel openers completely reversed ATP inhibition of K flux in both systems. In liposomes, ATP-inhibited K flux was restored by diazoxide (K = 0.4 μM), cromakalim (K = 1 μM), and two developmental cromakalim analogues, EMD60480 and EMD57970 (K = 6 nM). Similar K values were observed in intact mitochondria. These potencies are well within the range observed with plasma membrane K channels. We also compared the potencies of these K channel openers on the plasma membrane K channel purified from beef heart myocytes. The K channel from cardiac mitochondria is 2000-fold more sensitive to diazoxide than the channel from cardiac sarcolemma, indicating that two distinct receptor subtypes coexist within the myocyte. We suggest that the mitochondrial K channel is an important intracellular receptor that should be taken into account in considering the pharmacology of K channel openers.

Overview of Molecular Relationships in the Voltage-Gated Ion Channel Superfamily

Complex multicellular organisms require rapid and accurate transmission of information among cells and tissues and tight coordination of distant functions. In vertebrates, electrical signals and the resulting intracellular calcium transients control contraction of muscle, secretion of hormones,

Multipass membrane protein structure prediction using Rosetta.

We describe the adaptation of the Rosetta de novo structure prediction method for prediction of helical transmembrane protein structures. The membrane environment is modeled by embedding the protein chain into a model membrane represented by parallel planes defining hydrophobic, interface, and polar membrane layers for each energy evaluation. The optimal embedding is determined by maximizing the exposure of surface hydrophobic residues within the membrane and minimizing hydrophobic exposure outside of the membrane. Protein conformations are built up using the Rosetta fragment assembly method and evaluated using a new membrane‐specific version of the Rosetta low‐resolution energy function in which residue–residue and residue–environment interactions are functions of the membrane layer in addition to amino acid identity, distance, and density. We find that lower energy and more native‐like structures are achieved by sequential addition of helices to a growing chain, which may mimic some aspects of helical protein biogenesis after translocation, rather than folding the whole chain simultaneously as in the Rosetta soluble protein prediction method. In tests on 12 membrane proteins for which the structure is known, between 51 and 145 residues were predicted with root‐mean‐square deviation <4 Å from the native structure. Proteins 2006.

Molecular Determinants of Voltage-dependent Gating and Binding of Pore-blocking Drugs in Transmembrane Segment IIIS6 of the Na+ Channel α Subunit

Mutations of amino acid residues in the inner two-thirds of the S6 segment in domain III of the rat brain type IIA Na+ channel (G1460A to I1473A) caused periodic positive and negative shifts in the voltage dependence of activation, consistent with an α-helix having one face on which mutations to alanine oppose activation. Mutations in the outer one-third of the IIIS6 segment all favored activation. Mutations in the inner half of IIIS6 had strong effects on the voltage dependence of inactivation from closed states without effect on open-state inactivation. Only three mutations had strong effects on block by local anesthetics and anticonvulsants. Mutations L1465A and I1469A decreased affinity of inactivated Na+ channels up to 8-fold for the anticonvulsant lamotrigine and its congeners 227c89, 4030w92, and 619c89 as well as for the local anesthetic etidocaine. N1466A decreased affinity of inactivated Na+ channels for the anticonvulsant 4030w92 and etidocaine by 3- and 8-fold, respectively, but had no effect on affinity of the other tested compounds. Leu-1465, Asn-1466, and Ile-1469 are located on one side of the IIIS6 helix, and mutation of each caused a positive shift in the voltage dependence of activation. Evidently, these amino acid residues face the lumen of the pore, contribute to formation of the high-affinity receptor site for pore-blocking drugs, and are involved in voltage-dependent activation and coupling to closed-state inactivation.

Role of Amino Acid Residues in Transmembrane Segments IS6 and IIS6 of the Na+ Channel α Subunit in Voltage-dependent Gating and Drug Block

Alanine-scanning mutagenesis of transmembrane segments IS6 and IIS6 of the rat brain Nav1.2 channel α subunit identified mutations N418A in IS6 and L975A in IIS6 as causing strong positive shifts in the voltage dependence of activation. In contrast, mutations V424A in IS6 and L983A in IIS6 caused strong negative shifts. Most IS6 mutations opposed inactivation from closed states, but most IIS6 mutations favored such inactivation. Mutations L421C and L983A near the intracellular ends of IS6 and IIS6, respectively, exhibited significant sustained Na+ currents at the end of 30-ms depolarizations, indicating a role for these residues in Na+ channel fast inactivation. These residues, in combination with residues at the intracellular end of IVS6, are well situated to form an inactivation gate receptor. Mutation I409A in IS6 reduced the affinity of the local anesthetic etidocaine for the inactivated state by 6-fold, and mutations I409A and N418A reduced use-dependent block by etidocaine. No IS6 or IIS6 mutations studied affected inactivated-state affinity or use-dependent block by the neuroprotective drug sipatrigine (compound 619C89). These results suggest that the local anesthetic receptor site is formed primarily by residues in segments IIIS6 and IVS6 with the contribution of a single amino acid in segment IS6.

Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C‐terminal domain

Voltage‐gated Ca2+ channels of the CaV1 family initiate excitation–contraction coupling in cardiac, smooth, and skeletal muscle and are primary targets for regulation by the sympathetic nervous system in the ‘fight‐or‐flight’ response. In the heart, activation of β‐adrenergic receptors greatly increases the L‐type Ca2+ current through CaV1.2 channels, which requires phosphorylation by cyclic AMP‐dependent protein kinase (PKA) anchored via an A‐kinase anchoring protein (AKAP15). Surprisingly, the site of interaction of PKA and AKAP15 lies in the distal C‐terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the proteolytically cleaved distal C‐terminal domain forms a specific molecular complex with the truncated α1 subunit 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 membrane potentials. Ab initio structural modelling and site‐directed mutagenesis revealed a binding interaction between a pair of arginine residues in a predicted α‐helix in the proximal C‐terminal domain and a set of three negatively charged amino acid residues in a predicted helix–loop–helix bundle in the distal C‐terminal domain. Disruption of this interaction by mutation abolished the inhibitory effects of the distal C‐terminus on CaV1.2 channel function. These results provide the first functional characterization of this autoinhibitory complex, which may be a major form of the CaV1 family Ca2+ channels in cardiac and skeletal muscle cells, and reveal a unique ion channel regulatory mechanism in which proteolytic processing produces a more effective autoinhibitor of CaV1.2 channel function.

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Voltage-dependent gating of ion channels is essential for electrical signaling in excitable cells, but the structural basis for voltage sensor function is unknown. We constructed high-resolution structural models of resting, intermediate, and activated states of the voltage-sensing domain of the bacterial sodium channel NaChBac using the Rosetta modeling method, crystal structures of related channels, and experimental data showing state-dependent interactions between the gating charge-carrying arginines in the S4 segment and negatively charged residues in neighboring transmembrane segments. The resulting structural models illustrate a network of ionic and hydrogen-bonding interactions that are made sequentially by the gating charges as they move out under the influence of the electric field. The S4 segment slides 6–8 Å outward through a narrow groove formed by the S1, S2, and S3 segments, rotates ∼30°, and tilts sideways at a pivot point formed by a highly conserved hydrophobic region near the middle of the voltage sensor. The S4 segment has a 310-helical conformation in the narrow inner gating pore, which allows linear movement of the gating charges across the inner one-half of the membrane. Conformational changes of the intracellular one-half of S4 during activation are rigidly coupled to lateral movement of the S4–S5 linker, which could induce movement of the S5 and S6 segments and open the intracellular gate of the pore. We confirmed the validity of these structural models by comparing with a high-resolution structure of a NaChBac homolog and showing predicted molecular interactions of hydrophobic residues in the S4 segment in disulfide-locking studies.

A gating hinge in Na+ channels; a molecular switch for electrical signaling.

Voltage-gated sodium channels are members of a large family with similar pore structures. The mechanism of opening and closing is unknown, but structural studies suggest gating via bending of the inner pore helix at a glycine hinge. Here we provide functional evidence for this gating model for the bacterial sodium channel NaChBac. Mutation of glycine 219 to proline, which would strongly favor bending of the alpha helix, greatly enhances activation by shifting its voltage dependence -51 mV and slowing deactivation by 2000-fold. The mutation also slows voltage-dependent inactivation by 1200-fold. The effects are specific because substitutions of proline at neighboring positions and substitutions of other amino acids at position 219 have much smaller functional effects. Our results fit a model in which concerted bending at glycine 219 in the S6 segments of NaChBac serves as a gating hinge. This gating motion may be conserved in most members of this large ion channel protein family.

An emerging consensus on voltage-dependent gating from computational modeling and molecular dynamics simulations.

Developing an understanding of the mechanism of voltage-gated ion channels in molecular terms requires knowledge of the structure of the active and resting conformations. Although the active-state conformation is known from x-ray structures, an atomic resolution structure of a voltage-dependent ion channel in the resting state is not currently available. This has motivated various efforts at using computational modeling methods and molecular dynamics (MD) simulations to provide the missing information. A comparison of recent computational results reveals an emerging consensus on voltage-dependent gating from computational modeling and MD simulations. This progress is highlighted in the broad context of preexisting work about voltage-gated channels.