Annette Hohaus

The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton

Ahnak is a ubiquitously expressed giant protein of 5643 amino acids implicated in cell differentiation and signal transduction. In a recent study, we demonstrated the association of ahnak with the regulatory β2 subunit of the cardiac L‐type Ca2+ channel. Here we identify the most carboxyl‐terminal ahnak region (aa 5262–5643) to interact with recombinant β2a as well as with β2 and β1a isoforms of native muscle Ca2+ channels using a panel of GST fusion proteins. Equilibrium sedimentation analysis revealed Kd values of 55 ± 11 nM and 328 ± 24 nM for carboxyl‐terminal (aa 195–606) and amino‐terminal (aa 1–200) truncates of the β2a subunit, respectively. The same carboxylterminal ahnak region (aa 5262–5643) bound to G‐actin and cosedimented with F‐actin. Confocal microscopy of human left ventricular tissue localized the carboxylterminal ahnak portion to the sarcolemma including the T‐tubular system and the intercalated disks of cardiomyocytes. These results suggest that ahnak provides a structural basis for the subsarcolemmal cytoarchitecture and confers the regulatory role of the actin‐based cytoskeleton to the L‐type Ca2+ channel.—Hohaus, A., Person, V., Behlke, J., Schaper, J., Morano, I., Haase, H. The carboxyl‐terminal region of ahnak provides a link between cardiac L‐type Ca2+ channels and the actin‐based cytoskeleton. FASEB J. 16, 1205–1216 (2002)

Valerenic acid potentiates and inhibits GABAA 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

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 IC50 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.

Signaling from β-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 ~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 β2 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 β‐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 β subunit. We hypothesize that both phosphorylation of pp700 and its coupling to the β subunit play a physiological role in cardiac β ‐adrenergic signal transduction. Haase, H., Podzuweit, T., Lutsch, G., Hohaus, A., Kostka, S., Lindschau, C., Kott, M., Kraft, R., Morano, I. Signaling from β‐adrenoceptor to L‐type calcium channel: identification of a novel cardiac protein kinase A target that has similarities to AHNAK. FASEB J. 13, 2161–2172 (1999)

Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the CaV1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553–7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the CaV1.2 channel. Substitution of Ile-781, the CaV1.2 residue corresponding to Ile-745 in CaV1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779–782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of CaV1.2. Moreover there may be a flexible center of helix bending at positions 779–782 of CaV1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.

Calcium Current in Rat Cardiomyocytes Is Modulated by the Carboxyl-terminal Ahnak Domain

Ahnak, a protein of 5643 amino acids, interacts with the regulatory β-subunit of cardiac calcium channels and with F-actin. Recently, we defined the binding sites among the protein partners in the carboxyl-terminal domain of ahnak. Here we further narrowed down the β2-interaction sites to the carboxyl-terminal 188 amino acids of ahnak by the recombinant ahnak protein fragments P3 (amino acids 5456-5556) and P4 (amino acids 5556-5643). The effects of these P3 and P4 fragments on the calcium current were investigated under whole-cell patch clamp conditions on rat ventricular cardiomyocytes. P4 but not P3 increased significantly the current amplitude by 22.7 ± 5% without affecting its voltage dependence. The slow component of calcium current inactivation was slowed down by both P3 and P4, whereas only P3 slowed significantly the fast one. The composite recombinant protein fragment P3-P4 induced similar modifications to the ones induced by each of the ahnak fragments. In the presence of carboxyl-terminal ahnak protein fragments, isoprenaline induced a similar relative increase in current amplitude and shift in current kinetics. The actin-stabilizing agents, phalloidin and jasplakinolide, did not modify the effects of these ahnak protein fragments on calcium current in control conditions nor in the presence of isoprenaline. Hence, our results suggest that the functional effects of P3, P4, and P3-P4 on calcium current are mediated by targeting the ahnak-β2-subunit interaction rather than by targeting the ahnak-F-actin interaction. More specifically they suggest that binding of the β2-subunit to the endogenous subsarcolemmal giant ahnak protein re-primes the α1C/β2-subunit interaction and that the ahnak-derived proteins relieve the β2-subunit from this inhibition.

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

We investigated the expression of α1 and β 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 β2 and β3 subunits were used for detection and quantification of α1, β2, and β3 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-β2 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 β2 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 β3 subunit isoform detected. Our results suggest that both α1 and β2 subunit expression is upregulated in HOCM in a coordinate manner.

Timothy mutation disrupts the link between activation and inactivation in Ca(V)1.2 protein.

The Timothy syndrome mutations G402S and G406R abolish inactivation of CaV1.2 and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into the consequences of the G402S mutation on structure and function of the channel, we systematically mutated the corresponding Gly-432 of the rabbit channel and applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished channel inactivation. Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in contrast, parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of Gly-432 in domain I. Mutations of Gly-432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points, thereby tightly sealing the activation gate of CaV1.2 in the closed state. Such a structural hypothesis is supported by changes in activation gating induced by mutations of the G/A/G/A residues. The structural implications for CaV1.2 activation and inactivation gating are discussed.

Probing the Architecture of an L-type Calcium Channel with a Charged Phenylalkylamine EVIDENCE FOR A WIDELY OPEN PORE AND DRUG TRAPPING

Voltage-gated calcium channels are in a closed conformation at rest and open temporarily when the membrane is depolarized. To gain insight into the molecular architecture of Cav1.2, we probed the closed and open conformations with the charged phenylalkylamine (-)devapamil ((-)qD888). To elucidate the access pathway of (-)D888 to its binding pocket from the intracellular side, we used mutations replacing a highly conserved Ile-781 by threonine/proline in the pore-lining segment IIS6 of Cav1.2 (1). The shifted channel gating of these mutants (by 30–40 mV in the hyperpolarizing direction) enabled us to evoke currents with identical kinetics at different potentials and thus investigate the effect of the membrane potentials on the drug access per se. We show here that under these conditions the development of channel block by (-)qD888 is not affected by the transmembrane voltage. Recovery from block at rest was, however, accelerated at more hyperpolarized voltages. These findings support the conclusion that Cav1.2 must be opening widely to enable free access of the charged (-)D888 molecule to its binding site, whereas drug dissociation from the closed channel conformation is restricted by bulky channel gates. The functional data indicating a location of a trapped (-)D888 molecule close to the central pore region are supported by a homology model illustrating that the closed Cav1.2 is able to accommodate a large cation such as (-)D888.

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.