Stanislav Berjukow

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.

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.

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

State dependent dissociation of HERG channel inhibitors

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 IC50 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.

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.

Sequence Differences between α1C and α1S Ca2+ Channel Subunits Reveal Structural Determinants of a Guarded and Modulated Benzothiazepine Receptor

The molecular basis of the Ca2+channel block by (+)-cis-diltiazem was studied in class A/l-type chimeras and mutant α1C-aCa2+ channels. Chimeras consisted of either rabbit heart (α1C-a) or carp skeletal muscle (α1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from α1C-awere efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (−)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel α1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 α1C-a to IVS6 of α1Ssignificantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a l-type α1subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of l-type Ca2+channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on l-type channels.