Publications (15)69.5 Total impact
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Article: Expression and 1,4-dihydropyridine-binding properties of brain L-type calcium channel isoforms.
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ABSTRACT: The L-type calcium channel (LTCC) isoforms Ca(v)1.2 and Ca(v)1.3 display similar 1,4-dihydropyridine (DHP) binding properties and are both expressed in mammalian brain. Recent work implicates Ca(v)1.3 channels as interesting drug targets, but no isoform-selective modulators exist. It is also unknown to what extent Ca(v)1.1 and Ca(v)1.4 contribute to L-type-specific DHP binding activity in brain. To address this question and to determine whether DHPs can discriminate between Ca(v)1.2 and Ca(v)1.3 binding pockets, we combined radioreceptor assays and quantitative polymerase chain reaction (qPCR). We bred double mutants (Ca(v)-DM) from mice expressing mutant Ca(v)1.2 channels [Ca(v)1.2DHP(-/-)] lacking high affinity for DHPs and from Ca(v)1.3 knockouts [Ca(v)1.3(-/-)]. (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) and Ca(v)-DM brains was reduced to 15.1 and 4.4% of wild type, respectively, indicating that Ca(v)1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Ca(v)1.1 and Ca(v)1.4 alpha(1) subunits comprised 0.08% of the LTCC transcripts in mouse whole brain, suggesting that they cannot account for the residual binding. Instead, this could be explained by low-affinity binding (127-fold K(d) increase) to the mutated Ca(v)1.2 channels. Inhibition of (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) (predominantly Ca(v)1.3) and wild-type (predominantly Ca(v)1.2) brain membranes by unlabeled DHPs revealed a 3- to 4-fold selectivity of nitrendipine and nifedipine for the Ca(v)1.2 binding pocket, a finding further confirmed with heterologously expressed channels. This suggests that small differences in their binding pockets may allow development of isoform-selective modulators for LTCCs and that, because of their very low expression, Ca(v)1.1 and Ca(v)1.4 are unlikely to serve as drug targets to treat CNS diseases.Molecular pharmacology 12/2008; 75(2):407-14. · 4.53 Impact Factor -
Article: Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.
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ABSTRACT: Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.Journal of Biological Chemistry 08/2008; 283(30):20733-44. · 4.77 Impact Factor -
Chapter: The Molecular Basis of Ca2+ Antagonist Drug Action-Recent Developments
02/2007: pages 262-280; -
Article: C-terminal splicing reveals intramolecular gating modulation in Ca<sub>V</sub>1.3 L-type Ca<sup>2+ </sup>channels
BMC Pharmacology. 01/2007; -
Article: C-terminal modulator controls Ca2+-dependent gating of Ca(v)1.4 L-type Ca2+ channels.
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ABSTRACT: Tonic neurotransmitter release at sensory cell ribbon synapses is mediated by calcium (Ca2+) influx through L-type voltage-gated Ca2+ channels. This tonic release requires the channels to inactivate slower than in other tissues. Ca(v)1.4 L-type voltage-gated Ca2+ channels (LTCCs) are found at high densities in photoreceptor terminals, and alpha1 subunit mutations cause human congenital stationary night blindness type-2 (CSNB2). Ca(v)1.4 voltage-dependent inactivation is slow and Ca2+-dependent inactivation (CDI) is absent. We show that removal of the last 55 or 122 (C122) C-terminal amino acid residues of the human alpha1 subunit restores calmodulin-dependent CDI and shifts voltage of half-maximal activation to more negative potentials. The C terminus must therefore form part of a mechanism that prevents calmodulin-dependent CDI of Ca(v)1.4 and controls voltage-dependent activation. Fluorescence resonance energy transfer experiments in living cells revealed binding of C122 to C-terminal motifs mediating CDI in other Ca2+ channels. The absence of this modulatory mechanism in the CSNB2 truncation mutant K1591X underlines its importance for normal retinal function in humans.Nature Neuroscience 10/2006; 9(9):1108-16. · 15.53 Impact Factor -
Article: Effects of congenital stationary night blindness type 2 mutations R508Q and L1364H on Cav1.4 L-type Ca2+ channel function and expression.
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ABSTRACT: At least 48 mutations in the CACNA1F gene encoding retinal Ca(v)1.4 L-type Ca(2+) channels have been linked to X-linked recessive congenital stationary night blindness type 2 (CSNB2). A large number of these are missense mutations encoding full-length alpha1-subunits that can potentially form functional channels. We have previously shown that such missense mutations can confer their phenotype by different pathological mechanisms, such as complete lack of alpha1 subunit protein expression or dramatic changes in channel gating. Here we investigated the functional consequences of CSNB2 missense mutations R508Q and L1364H. We found no (R508Q) or only minor (L1364H) changes in the gating properties of both mutants after heterologous expression in Xenopus laevis oocytes (at 20 degrees C). However, both mutants resulted in altered expression density of Ca(v)1.4 currents. When expressed in the mammalian cell line tsA-201, the current amplitude of L1364H channels was reduced when cells were grown at 30 degrees C and both mutations affected total alpha1 protein expression. This effect was temperature dependent. Our data provide evidence that, in contrast to previously characterized CSNB2 missense mutations, the clinical phenotype of R508Q and L1364H is unlikely to be explained by changes in channel gating. Instead, these mutations affect the protein expression of Ca(v)1.4 Ca(2+) channels.Journal of Neurochemistry 04/2006; 96(6):1648-58. · 4.06 Impact Factor -
Article: Effects of congenital stationary night blindness type 2 mutations R508Q and L1364H on Ca v 1.4�L-type Ca 2+ channel function and expression
Journal of Neurochemistry - J NEUROCHEM. 01/2006; 96(6):1648-1658. -
Article: A CACNA1F mutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation.
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ABSTRACT: Light stimuli produce graded hyperpolarizations of the photoreceptor plasma membrane and an associated decrease in a voltagegated calcium channel conductance that mediates release of glutamate neurotransmitter. The Ca(v)1.4 channel is thought to be involved in this process. The CACNA1F gene encodes the poreforming subunit of the Ca(v)1.4 channel and various mutations in CACNA1F cause X-linked incomplete congenital stationary night blindness (CSNB2). The molecular mechanism of the pathology underlying the CSNB2 phenotype remains to be established. Recent clinical investigations of a New Zealand family found a severe visual disorder that has some clinical similarities to, but is clearly distinct from, CSNB2. Here, we report investigations into the molecular mechanism of the pathology of this condition. Molecular genetic analyses identified a previously undescribed nucleotide substitution in CACNA1F that is predicted to encode an isoleucine to threonine substitution at CACNA1F residue 745. The I745T CACNA1F allele produced a remarkable approximately -30-mV shift in the voltage dependence of Ca(v)1.4 channel activation and significantly slower inactivation kinetics in an expression system. These findings imply that substitution of this wild-type residue in transmembrane segment IIS6 may have decreased the energy required to open the channel. Collectively, these findings suggest that a gain-of-function mechanism involving increased Ca(v)1.4 channel activity is likely to cause the unusual phenotype.Proceedings of the National Academy of Sciences 06/2005; 102(21):7553-8. · 9.68 Impact Factor -
Article: Congenital stationary night blindness type 2 mutations S229P, G369D, L1068P, and W1440X alter channel gating or functional expression of Ca(v)1.4 L-type Ca2+ channels.
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ABSTRACT: Mutations in the CACNA1F gene (voltage-dependent L-type calcium channel alpha1F subunit) encoding retinal Ca(v)1.4 L-type Ca2+ channels cause X-linked recessive congenital stationary night blindness type 2 (CSNB2). Many of them are predicted to yield nonfunctional channels. Complete loss of Ca(v)1.4 function is therefore regarded as a pathogenetic mechanism for the impaired signaling from photoreceptors to second-order retinal neurons. We investigated the functional consequences of CSNB2 missense mutations S229P, G369D, and L1068P and the C-terminal truncation mutant W1440X. After expression in Xenopus laevis oocytes or human embryonic kidney tsA-201 cells, inward Ca2+ current (I(Ca)) and inward Ba2+ current (I(Ba)) could be recorded from mutations G369D and L1068P. G369D shifted the half-maximal voltage for channel activation (V(0.5,act)) significantly to more negative potentials (>11 mV), slowed inactivation, and removed Ca2+-dependent inactivation. The L1068P mutant yielded currents only in the presence of the channel activator BayK8644. Currents (I(Ba)) inactivated faster than wild type (WT) and recovered more slowly from inactivation (I(Ba) and I(Ca)). No channel activity could be measured for mutants S229P and W1440X after oocyte expression. No W1440X alpha1 protein was detected after expression in tsA-201 cells, whereas S229P (as well as G369D and L1068P) alpha1 subunits were expressed at levels indistinguishable from WT (n = 3). Our data provide unequivocal evidence that CSNB2 missense mutations can induce severe changes in Ca(v)1.4 function, which may decrease (L1068P and S229P) or even increase (G369D) channel activity. The lower activation range of G369D can explain the reduced dynamic range of photoreceptor signaling. Moreover, we demonstrate that loss of channel function of one (L1068P) CSNB2 mutation can be rescued by a Ca2+ channel activator.Journal of Neuroscience 01/2005; 25(1):252-9. · 7.11 Impact Factor -
Article: Opposite effects of a single IIIS5 mutation on phenylalkylamine and dihydropyridine interaction with L-type Ca2+ channels.
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ABSTRACT: Replacement of L-type Ca(2+) channel alpha(1) subunit residue Thr-1066 in segment IIIS5 by a tyrosine residue conserved in the corresponding positions of non-L-type Ca(2+) channels eliminates high dihydropyridine sensitivity through a steric mechanism. To determine the effects of this mutation on phenylalkylamine interaction, we exploited the availability of Ca(v)1.2DHP(-/-) mice containing the T1066Y mutation. In contrast to dihydropyridines, increased protein-dependent binding of the phenylalkylamine (-)-[(3)H]devapamil occurred to Ca(v)1.2DHP(-/-) mouse brain microsomes. This effect could be attributed to an at least 2-fold increase in affinity as determined by saturation analysis and binding inhibition experiments. The latter also revealed a higher affinity for (-)-verapamil but not for (-)-gallopamil. The mutation caused a pronounced slowing of (-)-[(3)H]devapamil dissociation, indicating a stabilization of the drug-channel complex. The increased affinity of mutant channels was also evident in functional studies after heterologous expression of wild type and T1066Y channels in Xenopus laevis oocytes. 100 mum (-)-verapamil inhibited a significantly larger fraction of Ba(2+) inward current through mutant than through WT channels. Our results provide evidence that phenylalkylamines also interact with the IIIS5 helix and that the geometry of the IIIS5 helix affects the access and/or binding of different chemical classes of Ca(2+) channel blockers to their overlapping binding domains. Mutation of Thr-1066 to a non-L-type tyrosine residue can be exploited to differentially affect phenylalkylamine and dihydropyridine binding to L-type Ca(2+) channels.Journal of Biological Chemistry 01/2005; 279(53):55211-7. · 4.77 Impact Factor -
Article: L-type Ca2+ channels in Ca2+ channelopathies.
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ABSTRACT: Voltage-gated L-type Ca2+ channels (LTCCs) mediate depolarization-induced Ca2+ entry in electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells. In this review we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within pore-forming alpha1 subunits causing incomplete congenital stationary night blindness, malignant hyperthermia sensitivity or hypokalemic periodic paralysis. However, studies in mice revealed that LTCC dysfunction also contributes to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting molecular tools to elucidate the contribution of different LTCC isoforms to human diseases.Biochemical and Biophysical Research Communications 11/2004; 322(4):1341-6. · 2.48 Impact Factor -
Article: Cav1.3 (alpha1D) Ca2+ currents in neonatal outer hair cells of mice.
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ABSTRACT: Outer hair cells (OHC) serve as electromechanical amplifiers that guarantee the unique sensitivity and frequency selectivity of the mammalian cochlea. It is unknown whether the afferent fibres connected to adult OHCs are functional. If so, voltage-activated Ca2+ channels would be required for afferent synaptic transmission. In neonatal OHCs, Ca2+ channels seem to play a role in maturation since OHCs from Cav1.3-deficient (Cav1.3-/-) mice degenerate shortly after the onset of hearing. We therefore studied whole-cell Ca2+ currents in outer hair cells aged between postnatal day 1 (P1) and P8. OHCs showed a rapidly activating inward current that was 1.8 times larger with 10 mM Ba2+ as charge carrier (IBa) than with equimolar Ca2+ (ICa). IBa started activating at -50 mV with Vmax = -1.9 +/- 6.9 mV, V0.5 = -15.0 +/- 7.1 mV and k = 8.2 +/- 1.1 mV (n = 34). The peak IBa showed negligible inactivation (3.6 % after 300 ms) whereas the ICa (10 mM Ca2+) was inactivated by 50.7 %. OHC IBa was reduced by 33.5 +/- 10.3 % (n = 14) with 10 microM nifedipine and increased to 178.5 +/- 57.8 % (n = 14) by 5 microM Bay K 8644. A dose-response curve for nifedipine revealed an IC50 of 2.3 microM, a Hill coefficient of 2.7 and a maximum block of 36 %. Average IBa density in OHCs was 24.4 +/- 10.8 pA pF-1 (n = 105) which is only 38 % of the value in inner hair cells. Single cell RT-PCR revealed expression of Cav1.3 in OHCs. In OHCs from Cav1.3-/- mice, Ba2+ current density was reduced to 0.6 +/- 0.5 pA pF-1 (n = 9) indicating that > 97 % of the Ca2+ channel current in OHCs flows through Cav1.3.The Journal of Physiology 12/2003; 553(Pt 3):747-58. · 4.72 Impact Factor -
Article: Cav1.3 (α1D) Ca2+ Currents in Neonatal Outer Hair Cells of Mice
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ABSTRACT: Outer hair cells (OHC) serve as electromechanical amplifiers that guarantee the unique sensitivity and frequency selectivity of the mammalian cochlea. It is unknown whether the afferent fibres connected to adult OHCs are functional. If so, voltage-activated Ca2+ channels would be required for afferent synaptic transmission. In neonatal OHCs, Ca2+ channels seem to play a role in maturation since OHCs from Cav1.3-deficient (Cav1.3−/−) mice degenerate shortly after the onset of hearing. We therefore studied whole-cell Ca2+ currents in outer hair cells aged between postnatal day 1 (P1) and P8. OHCs showed a rapidly activating inward current that was 1.8 times larger with 10 mm Ba2+ as charge carrier (IBa) than with equimolar Ca2+ (ICa). IBa started activating at −50 mV with Vmax=−1.9 ± 6.9 mV, V0.5=−15.0 ± 7.1 mV and k= 8.2± 1.1 mV (n= 34). The peak IBa showed negligible inactivation (3.6 % after 300 ms) whereas the ICa (10 mm Ca2+) was inactivated by 50.7 %. OHC IBa was reduced by 33.5 ± 10.3 % (n= 14) with 10 μm nifedipine and increased to 178.5 ± 57.8 % (n= 14) by 5 μm Bay K 8644. A dose-response curve for nifedipine revealed an IC50 of 2.3 μm, a Hill coefficient of 2.7 and a maximum block of 36 %. Average IBa density in OHCs was 24.4 ± 10.8 pA pF−1 (n= 105) which is only 38 % of the value in inner hair cells. Single cell RT-PCR revealed expression of Cav1.3 in OHCs. In OHCs from Cav1.3−/− mice, Ba2+ current density was reduced to 0.6 ± 0.5 pA pF−1 (n= 9) indicating that > 97 % of the Ca2+ channel current in OHCs flows through Cav1.3.The Journal of Physiology 11/2003; 553(3):747 - 758. · 4.72 Impact Factor -
Article: Cav1.4alpha1 subunits can form slowly inactivating dihydropyridine-sensitive L-type Ca2+ channels lacking Ca2+-dependent inactivation.
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ABSTRACT: The neuronal L-type calcium channels (LTCCs) Cav1.2alpha1 and Cav1.3alpha1 are functionally distinct. Cav1.3alpha1 activates at lower voltages and inactivates more slowly than Cav1.2alpha1, making it suitable to support sustained L-type Ca2+ inward currents (ICa,L) and serve in pacemaker functions. We compared the biophysical and pharmacological properties of human retinal Cav1.4alpha1 using the whole-cell patch-clamp technique after heterologous expression in tsA-201 cells with other L-type alpha1 subunits. Cav1.4alpha1-mediated inward Ba2+ currents (IBa) required the coexpression of alpha2delta1 and beta3 or beta2a subunits and were detected in a lower proportion of transfected cells than Cav1.3alpha1. IBa activated at more negative voltages (5% activation threshold; -39mV; 15 mm Ba2+) than Cav1.2alpha1 and slightly more positive than Cav1.3alpha1. Voltage-dependent inactivation of IBa was slower than for Cav1.2alpha1 and Cav1.3alpha1( approximately 50% inactivation after 5 sec; alpha2delta1 + beta3 coexpression). Inactivation was not increased with Ca2+ as the charge carrier, indicating the absence of Ca2+-dependent inactivation. Cav1.4alpha1 exhibited voltage-dependent, G-protein-independent facilitation by strong depolarizing pulses. The dihydropyridine (DHP)-antagonist isradipine blocked Cav1.4alpha1 with approximately 15-fold lower sensitivity than Cav1.2alpha1 and in a voltage-dependent manner. Strong stimulation by the DHP BayK 8644 was found despite the substitution of an otherwise L-type channel-specific tyrosine residue in position 1414 (repeat IVS6) by a phenylalanine. Cav1.4alpha1 + alpha2delta1 + beta channel complexes can form LTCCs with intermediate DHP antagonist sensitivity lacking Ca2+-dependent inactivation. Their biophysical properties should enable them to contribute to sustained ICa,L at negative potentials, such as required for tonic neurotransmitter release in sensory cells and plateau potentials in spiking neurons.Journal of Neuroscience 08/2003; 23(14):6041-9. · 7.11 Impact Factor -
Article: Cav1.41 Subunits Can Form Slowly Inactivating Dihydropyridine-Sensitive L-Type Ca2 Channels Lacking Ca2Dependent Inactivation
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ABSTRACT: The neuronal L-type calcium channels (LTCCs) Cav1.21 and Cav1.31 are functionally distinct. Cav1.31 activates at lower voltages and inactivates more slowly than Cav1.21, making it suitable to support sustained L-type Ca 2 inward currents (ICa,L ) and serve in pace- maker functions. We compared the biophysical and pharmacological properties of human retinal Cav1.41 using the whole-cell patch- clamp technique after heterologous expression in tsA-201 cells with other L-type1 subunits. Cav1.41-mediated inward Ba 2 currents (IBa ) required the coexpression of 21 and 3o r2a subunits and were detected in a lower proportion of transfected cells than Cav1.31. IBa activated at more negative voltages (5% activation threshold; 39mV; 15 mM Ba 2 ) than Cav1.21 and slightly more positive than Cav1.31. Voltage-dependent inactivation of IBa was slower than for Cav1.21 and Cav1.3 1( 50% inactivation after 5 sec; 21 3 coexpression). Inactivation was not increased with Ca 2 as the charge carrier, indicating the absence of Ca 2-dependent inactivation. Cav1.41 exhibited voltage-dependent, G-protein-independent facilitation by strong depolarizing pulses. The dihydropyr- idine (DHP)-antagonist isradipine blocked Cav1.41 with15-fold lower sensitivity than Cav1.21 and in a voltage-dependent manner. Strong stimulation by the DHP BayK 8644 was found despite the substitution of an otherwise L-type channel-specific tyrosine residue in position 1414 (repeat IVS6) by a phenylalanine. Cav1.41 21 channel complexes can form LTCCs with intermediate DHP antagonist sensitivity lacking Ca 2-dependent inactivation. Their biophysical properties should enable them to contribute to sustained
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Institutions
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2005–2008
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Universität Innsbruck
- • Institut für Pharmazie
- • Abteilung für Pharmakognosie
Innsbruck, Tyrol, Austria
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