Calcium channel gating.

Tuned calcium entry through voltage-gated calcium channels is a key requirement for many cellular functions. This is ensured by channel gates which open during membrane depolarizations and seal the pore at rest. The gating process is determined by distinct sub-processes: movement of voltage-sensing domains (charged S4 segments) as well as opening and closure of S6 gates. Neutralization of S4 charges revealed that pore opening of CaV1.2 is triggered by a "gate releasing" movement of all four S4 segments with activation of IS4 (and IIIS4) being a rate-limiting stage. Segment IS4 additionally plays a crucial role in channel inactivation. Remarkably, S4 segments carrying only a single charged residue efficiently participate in gating. However, the complete set of S4 charges is required for stabilization of the open state. Voltage clamp fluorometry, the cryo-EM structure of a mammalian calcium channel, biophysical and pharmacological studies, and mathematical simulations have all contributed to a novel interpretation of the role of voltage sensors in channel opening, closure, and inactivation. We illustrate the role of the different methodologies in gating studies and discuss the key molecular events leading CaV channels to open and to close.

New potential binding determinant for hERG channel inhibitors.

Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K(+) current (IKr) and are essential for the repolarization of the cardiac action potential. hERG1 inhibition by structurally diverse drugs may lead to life threatening arrhythmia. Putative binding determinants of hERG1 channel blockers include T623, S624 and V625 on the pore helix, and residues G648, Y652 and F656, located on segment S6. We and others have previously hypothesized that additional binding determinants may be located on helix S5, which is in close contact with the S6 segments. In order to test this hypothesis, we performed a detailed investigation combining ionic current measurements with two-microelectrode voltage clamp and molecular modeling techniques. We identified a novel aromatic high affinity binding determinant for blockers located in helix S5, F557, which is equally potent as Y652. Modeling supports a direct interaction with the outer pore helix.

Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site-directed mutagenesis.

BACKGROUND AND PURPOSE: ß2/3-subunit-selective modulation of GABAA receptors by valerenic acid (VA) is determined by the presence of transmembrane residue ß2/3N265. Currently, it is not known whether ß2/3N265 is part of VA's binding pocket or is involved in the transduction pathway of VA's action. The aim of this study was to clarify the localization of VA's binding pocket on GABAA receptors. EXPERIMENTAL APPROACH: Docking and a structure-based three-dimensional pharmacophore were employed to identify candidate amino acid residues that are likely to interact with VA. Selected amino acid residues were mutated, and VA-induced modulation of the resulting GABAA receptors expressed in Xenopus oocytes was analysed. KEY RESULTS: A binding pocket for VA at the ß(+) /α(-) interface encompassing amino acid ß3N265 was predicted. Mutational analysis of suggested amino acid residues revealed a complete loss of VA's activity on ß3M286W channels as well as significantly decreased efficacy and potency of VA on ß3N265S and ß3F289S receptors. In addition, reduced efficacy of VA-induced IGABA enhancement was also observed for α1M235W, ß3R269A and ß3M286A constructs. CONCLUSIONS AND IMPLICATIONS: Our data suggest that amino acid residues ß3N265, ß3F289, ß3M286, ß3R269 in the ß3 subunit, at or near the etomidate/propofol binding site(s), form part of a VA binding pocket. The identification of the binding pocket for VA is essential for elucidating its pharmacological effects and might also help to develop new selective GABAA receptor ligands.

Methods for quantification of pore-voltage sensor interaction in Ca(V)1.2.

Voltage sensors (VSs) initiate the pore opening and closure in voltage-gated ion channels. Here, we propose a technique for estimation of the equilibrium constant of the up- and downward VS movements and rate constants of pore transitions from macroscopic current kinetics. Bell-shaped voltage dependence of the activation/deactivation time constants and Bolzmann distributions of CaV1.2 activation were analyzed in terms of a circular four-state (rest, activated, open, deactivated) channel model: both dependencies uniquely constrain the model parameters. Neutralization of gating charges in IS4 or IIS4 only slightly affects the equilibrium constant of VS transition while affecting simultaneously the rate constants of pore opening and closure. The application of our technique revealed that pore mutations on IS6-IVS6 segments induce pronounced shifts of the VS equilibrium between the resting (down) and activated (up) position. Analyzing a channelopathy mutation highlighted that the leftward shift of the activation curve induced by I781T on IIS6 is only partially (35 %) caused by a destabilization of the channel pore but predominantly (65 %) by a shifted VS equilibrium towards activation. The algorithm proposed for CaV1.2 may be applicable for calculating rate constants from macroscopic current kinetics in other voltage-gated ion channels.

Interaction of diltiazem with an intracellularly accessible binding site on Ca(V)1.2.

BACKGROUND AND PURPOSE: Diltiazem inhibits Ca(V)1.2 channels and is widely used in clinical practice to treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α1 subunit of Ca(V)1.2. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane-impermeable quaternary derivative d-cis-diltiazem (qDil) and mutant α1 subunits. EXPERIMENTAL APPROACH: Ca(V)1.2 composed of α1, α2-δ and ß2a subunits were expressed in tsA-201 cells and barium currents through Ca(V)1.2 channels were recorded using the patch clamp method in the whole cell configuration. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion). KEY RESULTS: Quaternary derivative d-cis-diltiazem inhibited Ca(V)1.2 when applied to the intracellular side of the membrane in a use-dependent manner (59 ± 4% at 300 µM) and induced only a low level of tonic (non-use-dependent) block (16 ± 2% at 300 µM) when applied to the extracellular side of the membrane. Mutations in IIIS6 and IVS6 that have previously been shown to reduce the sensitivity of Ca(V)1.2 to tertiary diltiazem also had reduced sensitivity to intracellularly applied qDil. CONCLUSION AND IMPLICATIONS: The data show that use-dependent block of in Ca(V)1.2 by diltiazem occurs by interaction with a binding site accessible via a hydrophilic route from the intracellular side of the membrane.

Estimating the efficiency of benzodiazepines on GABA(A) receptors comprising gamma1 or gamma2 subunits.

BACKGROUND AND PURPOSE: Heterologous expression of alpha1, beta2 and gamma2S(gamma1) subunits produces a mixed population of GABA(A) receptors containing alpha1beta2 or alpha1beta2gamma2S(gamma1) subunits. GABA sensitivity (lower in receptors containing gamma1 or gamma2S subunits) and the potentiation of GABA-activated chloride currents (I(GABA)) by benzodiazepines (BZDs) are dependent on gamma2S(gamma1) incorporation. A variable gamma subunit incorporation may affect the estimation of I(GABA) potentiation by BZDs. We propose an approach for estimation of BZD efficiency that accounts for mixed population of alpha1beta2 and alpha1beta2gamma2S(gamma1) receptors. EXPERIMENTAL APPROACH: We investigated the relation between GABA sensitivity (EC50) and BZD modulation by analysing triazolam-, clotiazepam- and midazolam-induced potentiation of I(GABA) in Xenopus oocytes under two-microelectrode voltage clamp. KEY RESULTS: Plotting EC50 versus BZD-induced shifts of GABA concentration-response curves (DeltaEC50(BZD)) of oocytes injected with different amounts of alpha1, beta2 and gamma2S(gamma1) cRNA (1:1:1-1:1:10) revealed a linear regression between gamma2S(gamma1)-mediated reduction of GABA sensitivity (EC50) and DeltaEC50(BZD). The slope factors of the regression were always higher for oocytes expressing alpha1beta2gamma1 subunit receptors (1.8 +/- 0.1 (triazolam), 1.6 +/- 0.1 (clotiazepam), 2.3 +/- 0.2 (midazolam)) than for oocytes expressing alpha1beta2gamma2S receptors (1.4 +/- 0.1 (triazolam), 1.4 +/- 0.1 (clotiazepam), 1.3 +/- 0.1 (midazolam)). Mutant GABA(A) receptors (alpha1beta2-R207Cgamma2S) with lower GABA sensitivity showed higher drug efficiencies (slope factors=1.1 +/- 0.1 (triazolam), 1.1 +/- 0.1 (clotiazepam), 1.2 +/- 0.1 (midazolam)). CONCLUSIONS AND IMPLICATIONS: Regression analysis enabled the estimation of BZD efficiency when variable mixtures of alpha1beta2 and alpha1beta2gamma2S(gamma1) receptors are expressed and provided new insights into the gamma2S(gamma1) dependency of BZD action.

Valerenic acid potentiates and inhibits GABA(A) receptors: molecular mechanism and subunit specificity.

Valerian is a commonly used herbal medicinal product for the treatment of anxiety and insomnia. Here we report the stimulation of chloride currents through GABA(A) receptors (I(GABA)) by valerenic acid (VA), a constituent of Valerian. To analyse the molecular basis of VA action, we expressed GABA(A) receptors with 13 different subunit compositions in Xenopus oocytes and measured I(GABA) using the two-microelectrode voltage-clamp technique. We report a subtype-dependent stimulation of I(GABA) by VA. Only channels incorporating beta(2) or beta(3) subunits were stimulated by VA. Replacing beta(2/3) by beta(1) drastically reduced the sensitivity of the resulting GABA(A) channels. The stimulatory effect of VA on alpha(1)beta(2) receptors was substantially reduced by the point mutation beta(2N265S) (known to inhibit loreclezole action). Mutating the corresponding residue of beta(1) (beta(1S290N)) induced VA sensitivity in alpha(1)beta(1S290N) comparable to alpha(1)beta(2) receptors. Modulation of I(GABA) was not significantly dependent on incorporation of alpha(1), alpha(2), alpha(3) or alpha(5) subunits. VA displayed a significantly lower efficiency on channels incorporating alpha(4) subunits. I(GABA) modulation by VA was not gamma subunit dependent and not inhibited by flumazenil (1 microM). VA shifted the GABA concentration-effect curve towards lower GABA concentrations and elicited substantial currents through GABA(A) channels at > or = 30 microM. At higher concentrations (> or = 100 microM), VA and acetoxy-VA inhibit I(GABA). A possible open channel block mechanism is discussed. In summary, VA was identified as a subunit specific allosteric modulator of GABA(A) receptors that is likely to interact with the loreclezole binding pocket.

State dependent dissociation of HERG channel inhibitors.

BACKGROUND AND PURPOSE: Inhibition of HERG channels prolongs the ventricular action potential and the QT interval with the risk of torsade de pointes arrhythmias and sudden cardiac death. Many drugs induce greater inhibition of HERG channels when the cell membrane is depolarized frequently. The dependence of inhibition on the pulsing rate may yield different IC(50) values at different frequencies and thus affect the quantification of HERG channel block. We systematically compared the kinetics of HERG channel inhibition and recovery from block by 8 blockers at different frequencies. EXPERIMENTAL APPROACH: HERG channels were expressed heterologously in Xenopus oocytes and currents were measured with the two-electrode voltage clamp technique. KEY RESULTS: Frequency-dependent block was observed for amiodarone, cisapride, droperidol and haloperidol (group 1) whereas bepridil, domperidone, E-4031 and terfenadine (group 2) induced similar pulse-dependent block at all frequencies. With the group 1 compounds, HERG channels recovered from block in the presence of drug (recovery being voltage-dependent). No substantial recovery from block was observed with the second group of compounds. Washing out of bepridil, domperidone, E-4031 and terfenadine was substantially augmented by frequent pulsing. Mutation D540K in the HERG channel (which exhibits reopening at negative voltages) facilitated recovery from block by these compounds at -140 mV. CONCLUSION AND IMPLICATIONS: Drug molecules dissociate at different rates from open and closed HERG channels ('use-dependent' dissociation). Our data suggest that apparently 'trapped' drugs (group 2) dissociated from the open channel state whereas group 1 compounds dissociated from open and resting states.

Pharmacological properties of GABAA receptors containing gamma1 subunits.

GABA(A) receptors composed of alpha(1), beta(2), gamma(1) subunits are expressed in only a few areas of the brain and thus represent interesting drug targets. The pharmacological properties of this receptor subtype, however, are largely unknown. In the present study, we expressed alpha(1)beta(2)gamma(1)-GABA(A) receptors in Xenopus laevis oocytes and analyzed their modulation by 21 ligands from 12 structural classes making use of the two-microelectrode voltage-clamp method and a fast perfusion system. Modulation of GABA-induced chloride currents (I(GABA)) was studied at GABA concentrations eliciting 5 to 10% of the maximal response. Triazolam, clotiazepam, midazolam, 2-(4-methoxyphenyl)-2,3,5,6,7,8,9,10-octahydro-cyclohepta-(b)pyrazolo[4,3-d]pyridin-3-one (CGS 20625), 2-(4-chlorophenyl)-pyrazolo[4,3-c]quinolin-3-one (CGS 9896), diazepam, zolpidem, and bretazenil at 1 microM concentrations were able to significantly (>20%) enhance I(GABA) in alpha(1)beta(2)gamma(1) receptors. Methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, 3-methyl-6-[3-trifluoromethyl-phenyl]-1,2,4-triazolo[4,3-b]pyridazine (Cl 218,872), clobazam, flumazenil, 5-(6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)-3-methyl-[1,2,4]-oxadiazole (Ru 33203), 2-phenyl-4-(3-ethyl-piperidinyl)-quinoline (PK 9084), flurazepam, ethyl-7-methoxy-11,12,13,13a-tetrahydro-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c] [1,4]benzodiazepine-1-carboxylate (l-655,708), 2-(6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)-4-methyl-thiazole (Ru 33356), and 6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)phenylmethanone (Ru 32698) (1 microM each) had no significant effect, and flunitrazepam and 2-phenyl-4-(4-ethyl-piperidinyl)-quinoline (PK 8165) inhibited I(GABA). The most potent compounds triazolam, clotiazepam, midazolam, and CGS 20625 were investigated in more detail on alpha(1)beta(2)gamma(1) and alpha(1)beta(2)gamma(2S) receptors. The potency and efficiency of these compounds for modulating I(GABA) was smaller for alpha(1)beta(2)gamma(1) than for alpha(1)beta(2)gamma(2S) receptors, and their effects on alpha(1)beta(2)gamma(1) could not be blocked by flumazenil. CGS 20625 displayed the highest efficiency by enhancing at 100 microM I(GABA) (alpha(1)beta(2)gamma(2)) by 775 +/- 17% versus 526 +/- 14% I(GABA) (alpha(1)beta(2)gamma(1)) and 157 +/- 17% I(GABA) (alpha(1)beta(2)) (p < 0.05). These data provide new insight into the pharmacological properties of GABA(A) receptors containing gamma(1) subunits and may aid in the design of specific ligands for this receptor subtype.

Modulation of the smooth-muscle L-type Ca2+ channel alpha1 subunit (alpha1C-b) by the beta2a subunit: a peptide which inhibits binding of beta to the I-II linker of alpha1 induces functional uncoupling.

Modulation of the smooth-muscle Ca(2+) channel alpha1C-b subunit by the auxiliary beta2a subunit was studied in the HEK 293 (cell line from human embryonic kidney cells) expression system. In addition, we tested whether the alpha1-beta interaction in functional channels is sensitive to an 18-amino-acid synthetic peptide that corresponds to the sequence of the defined major interaction domain in the cytoplasmic I-II linker of alpha1C (AID-peptide). Ca(2+) channels derived by co-expression of alpha1C-b and beta2a subunits exhibited an about 3-fold higher open probability (P(o)) than alpha1C-b channels. High-P(o) gating of alpha1C-b.beta2a channels was associated with the occurrence of long-lasting channel openings [mean open time (tau)>10 ms] which were rarely observed in alpha1C-b channels. Modulation of fast gating by the beta2a subunit persisted in the cell-free, inside-out recording configuration. Biochemical experiments showed that the AID-peptide binds with appreciable affinity to beta2 subunits of native Ca(2+) channels. Binding of the beta2 protein to immobilized AID-peptide was specifically inhibited (K(i) of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-peptide (10 microM) to the cytoplasmic side of inside-out patches induced a substantial reduction of P(o) of alpha1C-b.beta2a channels. The scrambled control peptide failed to affect alpha1C-b. beta2a channels, and the AID-peptide (10 microM) did not modify alpha1C-b channel function in the absence of expressed beta2a subunit. Our results demonstrate that the beta2a subunit controls fast gating of alpha1C-b channels, and suggest the alpha1-beta interaction domain in the cytoplasmic I-II linker of alpha1C (AID) as a possible target of modulation of the channel. Moreover, our data are consistent with a model of alpha1-beta interaction that is based on multiple interaction sites, including AID as a determinant of the affinity of the alpha1-beta interaction.

Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target possessing similarities to AHNAK.

A novel calcium channel-associated protein of approximately 700 kDa has been identified in mammalian cardiomyocytes that undergoes substantial cAMP-dependent protein kinase (PKA) phosphorylation. It was therefore designated as phosphoprotein 700 (pp700). The pp700 interacts specifically with the beta(2) subunit of cardiac L-type calcium channels as revealed by coprecipitation experiments using affinity-purified antibodies against different calcium channel subunits. It is surprising that amino acid sequence analysis of pig pp700 revealed homology to AHNAK-encoded protein, which was originally identified in human cell lines of neural crest origin as 700-kDa phosphoprotein. Cardiac AHNAK expression was assessed on mRNA level by reverse transcriptase-polymerase chain reaction. Sequence-directed antibodies raised against human AHNAK recognized pp700 in immunoblotting and immunoprecipitation experiments, confirming the homology between both proteins. Anti-AHNAK antibodies labeled preferentially the plasma membrane of cardiomyocytes in cryosections of rat cardiac tissue and isolated cardiomyocytes. Sarcolemmal pp700/AHNAK localization was not influenced by stimulation of either the PKA or the protein kinase C pathway. In back-phosphorylation studies with cardiac biopsies, we identified distinct pp700 pools. The membrane-associated fraction of pp700 underwent substantial in vivo phosphorylation on beta-adrenergic receptor stimulation by isoproterenol, whereas the cytoplasmic fraction of pp700 was not accessible to endogenous PKA. It is important that in vivo phosphorylation occurred in that pp700 fraction which coprecipitated with the calcium channel beta subunit. We hypothesize that both phosphorylation of pp700 and its coupling to the beta subunit play a physiological role in cardiac beta-adrenergic signal transduction. Haase, H., Podzuweit, T., Lutsch, G., Hohaus, A., Kostka, S., Lindschau, C., Kott, M., Kraft, R., Morano, I. Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target that has similarities to AHNAK.

Characterization of naturally occurring myosin heavy chain antisense mRNA in rat heart.

Analysis of mRNA by Northern blot and reverse transcription-polymerase chain reaction demonstrated the expression of sense and considerable amounts of naturally occurring antisense mRNA for beta-myosin heavy chain (MHC) and alpha-MHC in the neonatal rat heart: antisense MHC mRNA expression of alpha-MHC and beta-MHC was approximately half of the corresponding sense MHC mRNA expression. Using a computational approach, we could identify a reverse Pol II promoter in the beta-MHC gene. Both sense and antisense MHC mRNA demonstrated similar sizes of approximately 6,000 bp in the Northern blot. Alpha-MHC antisense mRNA consisted of approximately 3,700 bp of complementary exon sequences and beta-MHC consisted of approximately 2,700 bp, suggesting a higher probability of alpha-MHC mRNA dimerization. Hence, sense mRNA transcripts and protein of alpha-MHC should exist at different relative levels in the neonatal state. In fact, the relative proportion of alpha-MHC was 52.0 +/- 2.6% on the sense mRNA but only 36.3 +/- 1.8% on the protein level. Because of its high abundance in the heart, we suggest that in the neonatal heart naturally occurring antisense mRNA may play a role in the regulation of MHC expression and, therefore, in the control of the energetical and contractile behaviour of the heart.

Expression of calcium channel subunits in the normal and diseased human myocardium.

We investigated the expression of alpha1 and beta subunits of the L-type Ca2+ channel on the protein level in cardiac preparations from normal human heart ventricles and from the hypertrophied septum of patients with hypertrophic obstructive cardiomyopathy (HOCM). 1,4-Dihydropyridine (DHP) binding and immunorecognition by polyclonal antibodies directed against the C-terminal amino acid sequences of the beta2 and beta3 subunits were used for detection and quantification of alpha1, beta2, and beta3 subunits. Bmax of high-affinity DHP binding was 35 +/- 2 fmol/mg protein in HOCM and 20 +/- 2 fmol/mg protein in normal human hearts (P<0.05). In rabbit hearts the anti-beta2 subunit antibody immunoprecipitated 80% of the total amount of DHP-labeled Ca2+ channels present in the assay. Under identical experimental conditions 25% of labeled Ca2+ channels were recovered in the immunoprecipitates of both normal and HOCM ventricles. A similar partial immunoprecipitation was observed in pig hearts. Immunoblot analysis demonstrated that the beta2 subunit was associated with the DHP receptor/Ca2+ channel in cardiac muscle of rabbit, pig, and human heart. In neither of these purified cardiac Ca2+ channels was the beta3 subunit isoform detected. Our results suggest that both alpha1 and beta2 subunit expression is upregulated in HOCM in a coordinate manner.