Rainer Marksteiner

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.

Autologous myoblasts and fibroblasts for female stress incontinence: a 1-year follow-up in 123 patients

To assess the efficacy and safety of the application of autologous myoblasts and fibroblasts for treating female stress urinary incontinence (SUI) after a follow‐up of ≥1 year.

A comparative study of three different biomaterials in the engineering of skeletal muscle using a rat animal model

Defects caused by traumatic or postsurgical loss of muscle mass may result in severe impairments of the functionality of skeletal muscle. Tissue engineering represents a possible approach to replace the lost or defective muscle. The aim of this study was to compare the suitability of three different biomaterials as scaffolds for rat myoblasts, using a new animal model. PKH26-fluorescent-stained cultured rat myoblasts were either seeded onto polyglycolic acid meshes or, alternatively, suspended in alginate or in hyaluronic acid-hydrogels. In each of the eight Fisher CDF-344 rats, four capsule pouches were induced by subcutaneous implantation of four silicone sheets. After two weeks the silicone sheets were removed and myoblast-biomaterial-constructs were implanted in the preformed capsules. Specimens were harvested after four weeks and examined histologically by H&E-staining and fluorescence microscopy. All capsules were well-vascularized. Implanted myoblasts fused by forming multinucleated myotubes. This study demonstrates that myoblasts seeded onto different biomaterials can be successfully transplanted into preformed highly vascularized capsule pouches. Our animal model has paved the way for studies of myoblast-biomaterial transplantations into an ectopic non-muscular environment.

pADPRT-2: a novel mammalian polymerizing(ADP-ribosyl)transferase gene related to truncated pADPRT homologues in plants and Caenorhabditis elegans

Until recently, poly(ADP‐ribosyl)ation was supposed to be confined only to polymerizing(ADP‐ribosyl)transferase/(ADP‐ribose)polymerase (E.C. 2.4.2.30). Here, we present novel polymerizing(ADP‐ribosyl)transferase homologues from mouse and man that lack all of the N‐terminal DNA binding and BRCA1 C‐terminus domains and will be designated polymerizing(ADP‐ribosyl)transferase‐2 as distinguished from the classical polymerizing(ADP‐ribosyl)transferase (polymerizing(ADP‐ribosyl)transferase‐1). The murine polymerizing(ADP‐ribosyl)transferase‐2 gene shares three identical intron positions with its Caenorhabditis elegans (EMBL nucleotide sequence database Z47075) and one with the Arabidopsis thaliana homologue (‘APP’, GenBank database AF069298). Expression of the murine polymerizing(ADP‐ribosyl)transferase‐2 gene was elevated in spleen, thymus and testis and the corresponding poly(ADP‐ribosyl)ation activity might account for most of the residual poly(ADP‐ribosyl)ation observed in polymerizing(ADP‐ribosyl)transferase‐1−/− mice.

Stem cell therapy for urinary stress incontinence

1. Introduction and rationaleThe increasing incidence in urinary incontinence (UI)with advancing age is directly correlated to spontaneousapoptosis of the muscle cells of the rhabdosphincter, thestriated urethral sphincter. As life expectancy of the wholesociety is growing, significance of UI as a disease of theelderly will increase further. Age-dependent reduction in thenumber of striated muscle cells is now held the highestranking cause of urinary stress incontinence in elderlywomen and men (Strasser et al., 1999, 2000a,b).The technical availability of autologous satellite cellsobtained from skeletal muscle enabled the immediateapplication of muscle progenitor cells in the treatment ofUI by means of tissue engineering techniques in patients ofany age, including elderly (Strasser et al., 2004).This review focuses on the theoretical assumptions andexperimental findings on muscle derived stem cells (MDSC)justifying their straightforward application in urology. Onlyquite recently, the regenerative function of mammaliantissues has become understood as ubiquitous (Deans andMoseley, 2000; Moore, 2002). Not only rapidly renewingtissues like skin, bone marrow or blood were found topossess a stem cell reserve: even apparently quiescenttissues, like nerve tissue or heart muscle disclosed—duringcellular stress or tissue loss—a tissue inherent renewingpotential (Hanashima et al., 2004). Tissue renewal dependson the persistence of so-called adult stem cells or precursorsof mature cells.Moreover, not only acute processes like injury orintoxication activate stem cell directed tissue regeneration.Cellular aging, cell death (apoptosis and necrosis) andrenewal continue throughout life and through all tissues.Imbalances between cell death and renewal are now thoughtto occur during aging (when apoptosis supersedes renewingcapacity) and during malignant transformation (whenregular cell death is hindered by apoptosis inhibition)(Hanahan and Weinberg, 2000).According to this rationale, the higher prevalence ofUI among elderly persons of both sexes can beinterpreted as a symptom of increasingly poor andeventually failing tissue regeneration in the vesico-urethral apparatus.A direct correlation between age and the number ofapoptotic cells in the rhabdomyosphincter on the one hand,and impaired detrusor contractility and weak closurepressure caused by structural deficits of the rhabdosphincteron the other hand, suggest that stress incontinence ispredominantly associated with a dysfunction of the striatedurethral sphincter (Frauscher et al., 1998, Strasser et al.,1999).Yiou and co-workers have shown that the adultrhabdosphincter in mice regenerates after an injury bymeans of intrinsic satellite cells (Yiou et al., 2003). Thisfinding is interesting per se, because rhabdosphincter andstriated skeletal muscles have a different embryologicalorigin. Muscle cells of the external sphincter arethought to arise via transdifferentiation of urethral smoothmuscle cells (Borirakchanyavat et al., 1997)—and thus,

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.

Inactivation determinants in segment IIIS6 of Cav3.1

1 Low threshold, T‐type, Ca2+ channels of the Cav3 family display the fastest inactivation kinetics among all voltage‐gated Ca2+ channels. The molecular inactivation determinants of this channel family are largely unknown. Here we investigate whether segment IIIS6 plays a role in Cav3.1 inactivation as observed previously in high voltage‐activated Ca2+ channels. 2 Amino acids that are identical in IIIS6 segments of all Ca2+ channel subtypes were mutated to alanine (F1505A, F1506A, N1509A, F1511A, V1512A, F1519A, FV1511/1512AA). Additionally M1510 was mutated to isoleucine and alanine. 3 The kinetic properties of the mutants were analysed with the two‐microelectrode voltage‐clamp technique after expression in Xenopus oocytes. The time constant for the barium current (IBa) inactivation, τinact, of wild‐type channels at −20 mV was 9.5 ± 0.4 ms; the corresponding time constants of the mutants ranged from 9.2 ± 0.4 ms in V1512A to 45.7 ± 5.2 ms (4.8‐fold slowing) in M1510I. Recovery at −80 mV was most significantly slowed by V1512A and accelerated by F1511A. 4 We conclude that amino acids M1510, F1511 and V1512 corresponding to previously identified inactivation determinants in IIIS6 of Cav2.1 ( Hering et al. 1998 ) have a significant role in Cav3.1 inactivation. These data suggest common elements in the molecular architecture of the inactivation mechanism in high and low threshold Ca2+ channels.

Molecular Mechanism of Calcium Channel Block by Isradipine ROLE OF A DRUG-INDUCED INACTIVATED CHANNEL CONFORMATION

The role of the inactivated channel conformation in the molecular mechanism of Ca2+ channel block by the 1,4-dihydropyridine (DHP) (+)-isradipine was analyzed in L-type channel constructs (α1Lc; Berjukow, S., Gapp, F., Aczel, S., Sinnegger, M. J., Mitterdorfer, J., Glossmann, H., and Hering, S. (1999) J. Biol. Chem. 274, 6154–6160) and a DHP-sensitive class A Ca2+ channel mutant (α1A-DHP; Sinnegger, M. J., Wang, Z., Grabner, M., Hering, S., Striessnig, J., Glossmann, H., and Mitterdorfer, J. (1997)J. Biol. Chem. 272, 27686–27693) carrying the high affinity determinants of the DHP receptor site but inactivating at different rates. Ca2+ channel inactivation was modulated by coexpressing the α1A-DHP- or α1Lc-subunits in Xenopus oocytes with either the β2a- or the β1a-subunit and amino acid substitutions in L-type segment IVS6 (I1497A, I1498A, and V1504A). Contrary to a modulated receptor mechanism assuming high affinity DHP binding to the inactivated state we observed no clear correlation between steady state inactivation and Ca2+ channel block by (+)-isradipine: (i) a 3-fold larger fraction of α1A-DHP/β1achannels in steady state inactivation at −80 mV (compared with α1A-DHP/β2a) did not enhance the block by (+)-isradipine; (ii) different steady state inactivation of α1Lc mutants at −30 mV did not correlate with voltage-dependent channel block; and (iii) the midpoint-voltages of the inactivation curves of slowly inactivating L-type constructs and more rapidly inactivating α1Lc/β1a channels were shifted to a comparable extent to more hyperpolarized voltages. A kinetic analysis of (+)-isradipine interaction with different L-type channel constructs revealed a drug-induced inactivated state. Entry and recovery from drug-induced inactivation are modulated by intrinsic inactivation determinants, suggesting a synergism between intrinsic inactivation and DHP block.

Specific neurotrophin binding to leucine-rich motif peptides of TrkA and TrkB

The extracellular domains of the TrkA and TrkB neurotrophin receptors contain defined structural modules such as immunoglobulin‐like domains and leucine‐rich motifs (LRMs) [Schneider and Schweiger, Oncogene 6 (1991) 1807–1811]. Recently, the second LRM of TrkA was identified as a functional nerve growth factor (NGF) binding site [Windisch et al, J. Biol. Chem. (1995) in press]. A peptide corresponding to this region effectively bound NGF and blocked binding of NGF to the recombinant extracellular domain of TrkA. The corresponding TrkB peptide exhibited the same effects with respect to brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and neurotrophin‐4 (NT‐4), indicating that all three TrkB ligands utilize this same binding site. Isolated LRMs therefore embody independent functional entities.

On the fate of skeletal myoblasts in a cardiac environment: down-regulation of voltage-gated ion channels

We have analysed the voltage‐gated ion channels and fusion competence of skeletal muscle myoblasts labelled with green fluorescent protein (GFP) and the membrane dye PKH transplanted into the infarcted myocardium of syngenic rats. After cell transplantation the animals were killed and GFP+–PKH+ myoblasts enzymatically isolated for subsequent studies of ionic currents through voltage‐gated sodium, calcium and potassium channels. A down‐regulation of all three types of ion channels after engraftment was observed. The fraction of cells with calcium (68%) and sodium channels (65%) declined to zero within 24 h and 1 week, respectively. Down‐regulation of potassium currents (90% in control) occurred within 2 weeks to about 30%. Before injection myoblasts expressed predominantly transient outward potassium channels whereas after isolation from the myocardium exclusively rapid delayed rectifier channels. The currents recovered completely between 1 and 6 weeks under cell culture conditions. The down‐regulation of ion channels and changes in potassium current kinetics suggest that the environment provided by infarcted myocardium affects  expression of voltage‐gated ion channels of skeletal myoblasts.