SummaryType 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.
L-type Ca2+ channels are characterized by their unique sensitivity to organic Ca2+ channel modulators like the 1,4-dihydropyridines (DHPs). To identify molecular motifs mediating DHP sensitivity, we transferred this sensitivity from L-type Ca2+ channels to the DHP-insensitive class A brain Ca2+ channel, BI-2. Expression of chimeras revealed minimum sequence stretches conferring DHP sensitivity including segments IIIS5, IIIS6, and the connecting linker, as well as the IVS5-IVS6 linker plus segment IVS6. DHP agonist and antagonist effects are determined by different regions within the repeat IV motif. Sequence regions responsible for DHP sensitivity comprise only 9.4% of the overall primary structure of a DHP-sensitive alpha 1A/alpha 1S construct. This chimera fully exhibits the DHP sensitivity of channels formed by L-type alpha 1 subunits. In addition, it displays the electrophysiological properties of alpha 1A, as well as its sensitivity toward the peptide toxins omega-agatoxin IVA and omega-conotoxin MVIIC.
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.Changes in channel gating were observed for mutants T666M, V714A, and I1819L but not for R192Q. Ba 2؉ current (I Ba ) inactivation was slightly faster in mutants T666M and V714A than in wild type. The time course of recovery from channel inactivation was slower than in wild type in T666M and accelerated in V714A and I1819L. As a consequence, accumulation of channel inactivation during a train of 1-Hz pulses was more pronounced for mutant T666M and less pronounced for V714A and I1819A. Our data demonstrate that three of the four FHM mutations, located at the putative channel pore, alter inactivation gating and provide a pathophysiological basis for the postulated neuronal instability in patients with FHM.␣ 1A subunits, in a complex with a  and ␣ 2 ␦ subunit (1, 2), constitute the pore-forming subunit of neuronal voltage-gated P/Q-type Ca 2ϩ channels. This channel type is not only located on nerve cell bodies and dendrites but is also present in presynaptic terminals (3) where it controls depolarization-induced Ca 2ϩ influx tightly coupled to neurotransmitter release (4). Its gating properties are modulated by neurotransmitters (5, 6) and affected by  subunits in an isoform-specific manner (7,8). This suggests that a tight control of P/Q-type Ca 2ϩ channel activity is a prerequisite to fine tune its physiological function.Missense mutations in the gene encoding human ␣ 1A (CACNL1A4) have recently been found to segregate with patients suffering from familial hemiplegic migraine (FHM) 1 (9), an autosomal dominant disorder. Although FHM represents a rare form of migraine, a detailed analysis of the functional consequences of this channelopathy may provide insight into the pathophysiology of migraine. Mutations in the CACNL1A4 gene could also underly more common forms of migraine with and without aura (10).The pathophysiology of migraine remains to be fully understood and the mechanisms triggering an attack are unknown. Recent advances in brain imaging techniques (positron emission tomography and magnetic resonance spectroscopy) support a "primary neuronal theory" where attacks originate on the basis of a neuronal hyperexcitability of unknown origin (11)(12)(13)(14)(15). This may be the underlying cause of cortical spreading depression and hypoperfusion, phenomena associated with migraine attacks (12, 13). Neuronal instability within central pain-modulating serotoninergic systems could not only serve as a "brainstem generator" of attacks but also initiate the headache and the events of neurogenic inflammation in the trigeminovascular ...
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 voltagedependent). 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 À140 mV. Conclusion and implications: Drug molecules dissociate at different rates from open and closed HERG channels ('usedependent' 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.
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