Steffen Hering

Familial Hemiplegic Migraine Mutations Change α1ACa2+ Channel Kinetics

Missense mutations in the pore-forming human α1A subunit of neuronal P/Q-type Ca2+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 α1A subunit into the conserved rabbit α1A(R192Q, T666M, V714A, and I1819L) and investigated possible changes in channel function after functional expression of mutant subunits inXenopus laevis oocytes. Changes in channel gating were observed for mutants T666M, V714A, and I1819L but not for R192Q. Ba2+ 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.

Proliferation of aligned mammalian cells on laser-nanostructured polystyrene.

Biomaterial surface chemistry and nanoscale topography are important for many potential applications in medicine and biotechnology as they strongly influence cell function, adhesion and proliferation. In this work, we present periodic surface structures generated by linearly polarized KrF laser light (248 nm) on polystyrene (PS) foils. These structures have a periodicity of 200-430 nm and a depth of 30-100 nm, depending on the angle of incidence of the laser beam. The changes in surface topography and chemistry were analysed by atomic force microscopy (AFM), advancing water contact-angle measurements, Fourier-transform infrared spectroscopy using an attenuated total reflection device (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). We show that the surface laser modification results in a significantly enhanced adhesion and proliferation of human embryonic kidney cells (HEK-293) compared to the unmodified polymer foil. Furthermore, we report on the alignment of HEK-293 cells, Chinese hamster ovary (CHO-K1) cells and skeletal myoblasts along the direction of the structures. The results indicate that the presence of nanostructures on the substrates can guide cell alignment along definite directions, and more importantly, in our opinion, that this alignment is only observed when the periodicity is above a critical periodicity value that is cell-type specific.

Isoform-specific regulation of mood behavior and pancreatic beta cell and cardiovascular function by L-type Ca 2+ channels.

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 beta 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 cardiovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca(v)1.2 alpha 1 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 beta 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 depression-like 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.

Transfer of 1,4-Dihydropyridine Sensitivity from L-Type to Class A (BI) Calcium Channels

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.

Molecular determinants of inactivation in voltage-gated Ca2+ channels

Evolution has created a large family of different classes of voltage‐gated Ca2+ channels and a variety of additional splice variants with different inactivation properties. Inactivation controls the amount of Ca2+ entry during an action potential and is, therefore, believed to play an important role in tissue‐specific Ca2+ signalling. Furthermore, mutations in a neuronal Ca2+ channel (Cav2.1) that are associated with the aetiology of neurological disorders such as familial hemiplegic migraine and ataxia cause significant changes in the process of channel inactivation. Ca2+ channels of a given subtype may inactivate by three different conformational changes: a fast and a slow voltage‐dependent inactivation process and in some channel types by an additional Ca2+‐dependent inactivation mechanism. Inactivation kinetics of Ca2+ channels are determined by the intrinsic properties of their pore‐forming α1‐subunits and by interactions with other channel subunits. This review focuses on structural determinants of Ca2+ channel inactivation in different parts of Ca2+ channel α1‐subunits, including pore‐forming transmembrane segments and loops, intracellular domain linkers and the carboxyl terminus. Inactivation is also affected by the interaction of the α1‐subunits with auxiliary β‐subunits and intracellular regulator proteins. The evidence shows that pore‐forming S6 segments and conformational changes in extra‐ (pore loop) and intracellular linkers connected to pore‐forming segments may play a principal role in the modulation of Ca2+ channel inactivation. Structural concepts of Ca2+ channel inactivation are discussed.

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.

Endogenous calcium channels in human embryonic kidney (HEK293) cells.

1 We have identified endogenous calcium channel currents in HEK293 cells. Whole cell endogenous currents (ISr‐HEK) were studied in single HEK293 cells with 10 mM strontium as the charge carrier by the patch clamp technique. The kinetic properties and pharmacological features of ISr‐HEK were characterized and compared with the properties of a heterologously expressed chimeric L‐type calcium channel construct. 2 ISr‐HEK activated on depolarization to voltages positive of −40 mV. It had transient current kinetics with a time to peak of 16 ± 1.4 ms (n = 7) and an inactivation times constant of 52 ± 5 ms (n = 7) at a test potential of 0 mV. The voltage for half maximal activation was −19.0 ± 1.5 mV (n = 7) and the voltage for half maximal steady‐state inactivation was −39.7 ± 2.3 mV (n = 7). 3 Block of ISr‐HEK by the dihydropyridine isradipine was not stereoselective; 1 μm (+) and (−)−isradipine inhibited the current by 30 ± 4% (n = 3) and 29 ± 2% (n = 4) respectively. (+)‐Isradipine and (−)−isradipine (10 μm) inhibited ISr‐HEK by 89 ± 4% (n = 5) and 88 ± 8% (n = 3) respectively. The 7‐bromo substituted (±)‐isradipine (VO2605, 10 μm) which is almost inactive on L‐type calcium channels also inhibited ISr‐HEK (83 ± 9%, n = 3) as was observed for 10 μm (−)−nimodipine (78 ± 6%, n = 5). Interestingly, 10 μm (±)‐Bay K 8644 (n = 5) had no effect on the current. ISr‐HEK was only slightly inhibited by the cone snail toxins ω‐CTx GVIA (1 μm, inhibition by 17 ± 3%, n = 4) and ω‐CTx MVIIC (1 μm, inhibition by 20 ± 3%, n = 4). The funnel web spider toxin ω‐Aga IVA (200 nM) inhibited ISr‐HEK by 19 ± 2%, n = 4). 4 In cells expressing ISr‐HEK, maximum inward current densities of 0.24 ± 0.03 pA/pF and 0.39 ± 0.7 pA/pF (at a test potential of −10 mV) were estimated in two different batches of HEK293 cells. The current density increased to 0.88 ± 0.18 pA/pF or 1.11 ± 0.2 pA/pF respectively, if the cells were cultured for 4 days in serum‐free medium. 5 Co‐expression of a chimeric L‐type calcium channel construct revealed that ISr‐HEK and L‐type calcium channel currents could be distinguished by their different voltage‐dependencies and current kinetics. The current density after heterologous expression of the L‐type α1 subunit chimera was estimated to be about ten times higher in serum containing medium (2.14 ± 0.45 pA/pF) than that of ISr‐HEK under the same conditions.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity

Summary Type 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.

Transfer of High Sensitivity for Benzothiazepines from L-type to Class A (BI) Calcium Channels

To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (α1S or α1C) to a weakly sensitive, nonstereoselective class A (α1A) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type α1 into the α1A subunit (I1804Y, S1808A, and M1811I) was sufficient to support a use-dependent block by diltiazem and by the phenylalkylamine (−)-gallopamil after expression in Xenopus oocytes. An additional mutation F1805M increased the sensitivity for (−)-gallopamil but not for diltiazem. Our data suggest that the receptor domains for diltiazem and gallopamil have common but not identical molecular determinants in transmembrane segment IVS6. These mutations also identified single amino acid residues in segment IVS6 that are important for class A channel inactivation.

Ca2+ channel block and inactivation: common molecular determinants

. Unexpectedly, thetransfer of the key amino acidsinvolved in sensitivity to DHPs,PAAs and BTZs results not only inthe transfer of the high-affinity drug-binding sites but also in the transferof a whole complex of propertiesthat characterizes the interaction ofthe drug with the native, donor L-type channels