Natural Science by Martina Brauns
Molecular pharmacology, 2009
The
The Journal of clinical investigation, 2004
Ca v 1.2 and Ca v 1.3 L-type Ca 2+ channels (LTCCs) are believed to underlie Ca 2+ currents in br... more Ca v 1.2 and Ca v 1.3 L-type Ca 2+ channels (LTCCs) are believed to underlie Ca 2+ currents in brain, pancreatic β cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause card iovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca v 1.2 α1 subunits (Ca v 1.2DHP -/-) without affecting function and expression. This allowed separation of the DHP effects of Ca v 1.2 from those of Ca v 1.3 and other LTCCs. DHP effects on pancreatic β cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Ca v 1.2DHP -/mice, which rules out a direct role of Ca v 1.3 for these physiological processes. Using Ca v 1.2DHP -/mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depressionlike behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Ca v 1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.
The Journal of biological chemistry, 1998
Missense mutations in the pore-forming human ␣ 1A subunit of neuronal P/Q-type Ca 2؉ channels are... more Missense mutations in the pore-forming human ␣ 1A subunit of neuronal P/Q-type Ca 2؉ channels are associated with familial hemiplegic migraine (FHM). The pathophysiological consequences of these mutations are unknown. We have introduced the four single mutations reported for the human ␣ 1A subunit into the conserved rabbit ␣ 1A (R192Q, T666M, V714A, and I1819L) and investigated possible changes in channel function after functional expression of mutant subunits in Xenopus laevis oocytes.
The Journal of biological chemistry, 1997
Pharmacological modulation by 1,4-dihydropyridines is a central feature of L-type calcium channel... more Pharmacological modulation by 1,4-dihydropyridines is a central feature of L-type calcium channels. Recently, eight L-type amino acid residues in transmembrane segments IIIS5, IIIS6, and IVS6 of the calcium channel ␣ 1 subunit were identified to substantially contribute to 1,4-dihydropyridine sensitivity. To determine whether these eight L-type residues (␣ 1C-a numbering) are sufficient to form a high affinity 1,4dihydropyridine binding site in a non-L-type calcium channel, we transferred them to the 1,4-dihydropyridine-insensitive ␣ 1A subunit using site-directed mutagenesis. 1,4-Dihydropyridine agonist and antagonist modulation of barium inward currents mediated by the mutant ␣ 1A subunits, coexpressed with ␣ 2 ␦ and  1a subunits in Xenopus laevis oocytes, was investigated with the two-microelectrode voltage clamp technique. The resulting mutant ␣ 1A-DHPi displayed low sensitivity for 1,4-dihydropyridines. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type ␣ 1 subunit previously cloned from Musca domestica body wall muscle led to the identification of Met 1188 (␣ 1C-a numbering) as an additional critical constituent of the L-type 1,4dihydropyridine binding domain. The introduction of this residue into ␣ 1A-DHPi restored full sensitivity for 1,4-dihydropyridines. It also transferred functional properties considered hallmarks of 1,4-dihydropyridine agonist and antagonist effects (i.e. stereoselectivity, voltage dependence of drug modulation, and agonistinduced shift in the voltage-dependence of activation). Our gain-of-function mutants provide an excellent model for future studies of the structure-activity relationship of 1,4-dihydropyridines to obtain critical structural information for the development of drugs for neuronal, non-L-type calcium channels. The abbreviations used are: DHP, 1,4-dihydropyridine; I Ba , barium inward current(s); V hϱ 1 ⁄2 , midpoint voltage of steady-state inactivation.
Biochemistry, 1995
The molecular determinants for Ca2+ modulation of dihydropyridine (DHP) binding to cardiac Ca2+ c... more The molecular determinants for Ca2+ modulation of dihydropyridine (DHP) binding to cardiac Ca2+ channels were identified by mutational neutralization of the glutamate residues that comprise the Ca2+ channel selectivity filter. The binding activity of the DHP (+)-[3H]isradipine, monitored after expression of wild-type and mutant alpha 1 subunits in COS-7 cells, was markedly reduced in four single mutants and a double mutant. Evidence for decreased Ca2+ affinity was obtained for two single mutants in kinetic and equilibrium binding studies. Mutational destabilization of Ca2+ binding resulted in a concomitant decrease of (+)-[3H]isradipine binding affinity. Recovery of (+)-[3H]isradipine binding activity by the allosteric modulator (+)-tetrandrine in two single mutants was associated with a recovery of Ca2+ and DHP binding kinetics to wild-type values. Our findings demonstrate that high-affinity DHP binding is dependent on Ca2+ coordination by glutamate residues which form the selectivity filter of the channel pore.
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Natural Science by Martina Brauns