FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2010481540> ?p ?o ?g. }

• W2010481540 endingPage "53" @default.
• W2010481540 startingPage "43" @default.
• W2010481540 abstract "L-type (CaV1.2) calcium channel antagonists play an important role in the treatment of cardiovascular disease. (R)-Roscovitine, a trisubstituted purine, has been shown to inhibit L-currents by slowing activation and enhancing inactivation. This study utilized molecular and pharmacological approaches to determine whether these effects result from (R)-roscovitine binding to a single site. Using the S enantiomer, we find that (S)-roscovitine enhances inactivation without affecting activation, which suggests multiple sites. This was further supported in studies using chimeric channels comprised of N- and L-channel domains. Those chimeras containing L-channel domains I and IV showed (R)-roscovitine-induced slowed activation like that of wild type L-channels, whereas chimeric channels containing L-channel domain I responded to (R)-roscovitine with enhanced inactivation. We conclude that (R)-roscovitine binds to distinct sites on L-type channels to slow activation and enhance inactivation. These sites appear to be unique from other calcium channel antagonist sites that reside within domains III and IV and are thus novel sites that could be exploited for future drug development. Trisubstituted purines could become a new class of drugs for the treatment of diseases related to hyperfunction of L-type channels, such as Torsades de Pointes. L-type (CaV1.2) calcium channel antagonists play an important role in the treatment of cardiovascular disease. (R)-Roscovitine, a trisubstituted purine, has been shown to inhibit L-currents by slowing activation and enhancing inactivation. This study utilized molecular and pharmacological approaches to determine whether these effects result from (R)-roscovitine binding to a single site. Using the S enantiomer, we find that (S)-roscovitine enhances inactivation without affecting activation, which suggests multiple sites. This was further supported in studies using chimeric channels comprised of N- and L-channel domains. Those chimeras containing L-channel domains I and IV showed (R)-roscovitine-induced slowed activation like that of wild type L-channels, whereas chimeric channels containing L-channel domain I responded to (R)-roscovitine with enhanced inactivation. We conclude that (R)-roscovitine binds to distinct sites on L-type channels to slow activation and enhance inactivation. These sites appear to be unique from other calcium channel antagonist sites that reside within domains III and IV and are thus novel sites that could be exploited for future drug development. Trisubstituted purines could become a new class of drugs for the treatment of diseases related to hyperfunction of L-type channels, such as Torsades de Pointes. Cardiac L-type (CaV1.2) channels are central to the regulation of a number of physiological processes (1Striessnig J. Hoda J.C. Koschak A. Zaghetto F. Müllner C. Sinnegger-Brauns M.J. Wild C. Watschinger K. Trockenbacher A. Pelster G. Biochem. Biophys. Res. Commun. 2004; 322: 1341-1346Crossref PubMed Scopus (40) Google Scholar, 2Bourinet E. Mangoni M.E. Nargeot J. J. Clin. Investig. 2004; 113: 1382-1384Crossref PubMed Scopus (15) Google Scholar). Activation of these channels in cardiac and smooth muscle generates the calcium influx that triggers calcium release from the sarcoplasmic reticulum (3Roden D.M. Balser J.R. George Jr., A.L. Anderson M.E. Annu. Rev. Physiol. 2002; 64: 431-475Crossref PubMed Scopus (228) Google Scholar) to induce contraction. In contrast, inactivation of these channels provides a negative feedback mechanism to limit calcium influx into the cardiac myocyte, which helps protect from excessive calcium influx that can lead to cardiac arrhythmias (3Roden D.M. Balser J.R. George Jr., A.L. Anderson M.E. Annu. Rev. Physiol. 2002; 64: 431-475Crossref PubMed Scopus (228) Google Scholar). L-channel antagonists are routinely used to treat cardiovascular diseases, such as hypertension and angina pectoris (4Glasser S.P. Neutel J.M. Gana T.J. Albert K.S. Am. J. Hypertens. 2003; 16: 51-58Crossref PubMed Scopus (44) Google Scholar, 5Bai R. Clin. Cardiol. 2005; 28: 343-348Crossref PubMed Scopus (2) Google Scholar, 6Elmslie K.S. J. Neurosci. Res. 2004; 75: 733-741Crossref PubMed Scopus (49) Google Scholar, 7Suzuki S. Ohtsuka S. Ishikawa K. Yamaguchi I. Hypertens. Res. 2003; 26: 193-199Crossref PubMed Scopus (10) Google Scholar, 8Thomas M.G. Sander G.E. Given M.B. Quiroz A.C. Roffidal L.E. Giles T.D. J. Clin. Pharmacol. 1990; 30: 24-28Crossref PubMed Scopus (2) Google Scholar). Therefore, drugs that inhibit L-channel function have high clinical relevance. Roscovitine is a 2,6,9-trisubstituted purine that was originally developed as a selective blocker of cyclin-dependent kinases (9Meijer L. Raymond E. Acc. Chem. Res. 2003; 36: 417-425Crossref PubMed Scopus (327) Google Scholar) and is currently undergoing phase II clinical trials as an anticancer drug (10Benson C. White J. De Bono J. O'Donnell A. Raynaud F. Cruickshank C. McGrath H. Walton M. Workman P. Kaye S. Cassidy J. Gianella-Borradori A. Judson I. Twelves C. Br. J. Cancer. 2007; 96: 29-37Crossref PubMed Scopus (220) Google Scholar). It has recently become apparent that roscovitine can affect voltage-dependent ion channels at clinically relevant concentrations (10–50 μm) (10Benson C. White J. De Bono J. O'Donnell A. Raynaud F. Cruickshank C. McGrath H. Walton M. Workman P. Kaye S. Cassidy J. Gianella-Borradori A. Judson I. Twelves C. Br. J. Cancer. 2007; 96: 29-37Crossref PubMed Scopus (220) Google Scholar, 11Vitali L. Yakisich J.S. Vita M.F. Fernandez A. Settembrini L. Siden A. Cruz M. Carminatti H. Casas O. Idoyaga Vargas V. Cancer Lett. 2002; 180: 7-12Crossref PubMed Scopus (20) Google Scholar, 12Raynaud F.I. Whittaker S.R. Fischer P.M. McClue S. Walton M.I. Barrie S.E. Garrett M.D. Rogers P. Clarke S.J. Kelland L.R. Valenti M. Brunton L. Eccles S. Lane D.P. Workman P. Clin. Cancer Res. 2005; 11: 4875-4887Crossref PubMed Scopus (110) Google Scholar, 13McClue S.J. Blake D. Clarke R. Cowan A. Cummings L. Fischer P.M. MacKenzie M. Melville J. Stewart K. Wang S. Zhelev N. Zheleva D. Lane D.P. Int. J. Cancer. 2002; 102: 463-468Crossref PubMed Scopus (326) Google Scholar). We (14Buraei Z. Anghelescu M. Elmslie K.S. Biophys. J. 2005; 89: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 15Buraei Z. Schofield G. Elmslie K.S. Neuropharmacology. 2007; 52: 883-894Crossref PubMed Scopus (45) Google Scholar) and others (16Yan Z. Chi P. Bibb J.A. Ryan T.A. Greengard P. J. Physiol. 2002; 540: 761-770Crossref PubMed Scopus (90) Google Scholar, 17Cho S. Meriney S.D. Eur. J. Neurosci. 2006; 23: 3200-3208Crossref PubMed Scopus (29) Google Scholar) have shown that (R)-roscovitine differentially affects voltage-dependent calcium channels. (R)-Roscovitine has two effects on CaV2 channels (N-type, P/Q-type, and R-type), which are a rapid onset agonist effect and a more slowly developing antagonist effect (14Buraei Z. Anghelescu M. Elmslie K.S. Biophys. J. 2005; 89: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 15Buraei Z. Schofield G. Elmslie K.S. Neuropharmacology. 2007; 52: 883-894Crossref PubMed Scopus (45) Google Scholar). The agonist effect results from (R)-roscovitine specifically binding to activated CaV2 channels to slow channel closing (14Buraei Z. Anghelescu M. Elmslie K.S. Biophys. J. 2005; 89: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 15Buraei Z. Schofield G. Elmslie K.S. Neuropharmacology. 2007; 52: 883-894Crossref PubMed Scopus (45) Google Scholar), which results in a significant enhancement of action potential induced calcium influx (14Buraei Z. Anghelescu M. Elmslie K.S. Biophys. J. 2005; 89: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The antagonist effect appears to result from (R)-roscovitine preferentially enhancing occupancy of a “resting” inactivated state to inhibit channel activity (18Buraei Z. Elmslie K.S. J. Neurochem. 2008; 105: 1450-1461Crossref PubMed Scopus (16) Google Scholar). Interestingly, the racemic variant (S)-roscovitine has been found to exhibit only an antagonist effect on N-channels, which is one result supporting unique binding sites for the agonist versus antagonist effects (14Buraei Z. Anghelescu M. Elmslie K.S. Biophys. J. 2005; 89: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 15Buraei Z. Schofield G. Elmslie K.S. Neuropharmacology. 2007; 52: 883-894Crossref PubMed Scopus (45) Google Scholar, 18Buraei Z. Elmslie K.S. J. Neurochem. 2008; 105: 1450-1461Crossref PubMed Scopus (16) Google Scholar). L-type channels are also inhibited by (R)-roscovitine, but by a unique mechanism relative to N-channels (19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar). L-channel inhibition results from slowed activation and enhanced open state voltage-dependent inactivation (VDI), 3The abbreviations used are: VDIvoltage-dependent inactivationHSDhonestly significant differenceL-DI, L-DII, L-DIII, and L-DIVL-channel domains I, II, III, and IV, respectivelyN-DI, N-DII, N-DIII, and N-DIVN-channel domains I, II, III, and IV, respectivelyWTwild typeRRSAroscovitine receptor site mediating slowed activationRREIroscovitine receptor site mediating enhanced inactivation. but the resting inactivated state is not affected. These two effects were characterized by approximately equal EC50 values (∼30 μm), which suggested a single binding site. However, the Hill coefficient for (R)-roscovitine-induced slowed activation was ∼1, whereas that for enhanced inactivation was >2, which could result from multiple binding sites (19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar). Intracellularly applied (R)-roscovitine failed to affect L-channel activity, which supported an extracellularly exposed binding site(s) mediating both effects. The differential effect of (R)-roscovitine on N-type versus L-type channels provides an opportunity to localize the L-channel binding site(s) by making chimeric channels. In addition, the differential effect of racemic roscovitine variants on N-channels led us to investigate if the L-channel site(s) also showed chiral specificity. voltage-dependent inactivation honestly significant difference L-channel domains I, II, III, and IV, respectively N-channel domains I, II, III, and IV, respectively wild type roscovitine receptor site mediating slowed activation roscovitine receptor site mediating enhanced inactivation. We found that (S)-roscovitine enhanced L-channel VDI without slowing activation, which supports separate binding sites for these effects. In addition, the chimeric channels showed that the L-channel domain I (L-DI) is both necessary and sufficient for the (R)-roscovitine-induced enhancement of VDI, but slowed activation requires both L-DI and L-DIV. These results reveal novel sites on the L-type calcium channel that can be exploited for the development of drugs that can specifically target the activation or inactivation function. Chimeric calcium channels (Fig. 1) were constructed using cDNAs encoding the rabbit cardiac L-channel (GenBankTM number X15539) and rat neuronal N-channel (GenBankTM number AF055477; generously provided by Dr. Lucie Parent) α1 subunits. Briefly, N-channel domains were amplified by PCR, subcloned into pCR-Blunt II-TOPO vectors (Invitrogen), excised by restriction enzyme digestion, and subcloned in frame into an engineered L-channel. The sequences of the wild type (WT) and mutant channels were aligned using Vector NTI (Invitrogen), and domain boundaries were placed in regions of high amino acid sequence homology. The engineered L-channel was generated by introducing unique AgeI and NotI sites into the intracellular linkers between the II/III (nucleotide 2751) and III/IV (nucleotide 3680) domains, respectively, using silent mutagenesis via the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). A unique XbaI site was introduced at nucleotide 4633 in the C terminus of the engineered channel using a similar strategy. The overall integrity of the engineered L-channel and each chimera was confirmed by qualitative restriction enzyme digests, DNA sequence analyses, Western blot analysis, and patch clamp electrophysiology . The chimeric channels were constructed as described below. For NNLL, L-DI and L-DII (Met1–Asn917) were replaced by N-DI and N-DII (Met1–Asn1134). N-DI and N-DII were amplified from full-length CaV2.2 and subcloned into the HindIII/AgeI sites of the engineered L-channel. For LLNN, L-DIII and L-DIV (Leu895–Leu2171) were replaced by N-DIII and N-DIV (Asp1112–Cys2333). The construction of this chimera did not utilize the engineered L-channel containing the introduced AgeI and NotI sites. Instead, a XbaI site at nucleotide 2678 was introduced into the L-channel, resulting in a missense mutation of a glutamate to arginine at position 894. For NLLL, L-DI (Met1–Cys519) was replaced by N-DI (Met1–Pro447). The insert for this chimera was constructed via overlap extension PCR. Briefly, N-DI and L-DII were amplified from full-length CaV2.2 and CaV1.2, respectively. The PCR products of these two domains contain a small region of overlap. They were then combined and used as templates in a second round of PCR. The final PCR product was subcloned into the HindIII/AgeI sites of the engineered L-channel. For LNLL, L-DII (Ala524–Val781) was replaced by N-DII (Ser452–Val909). This chimera was constructed using a strategy similar to that described for NLLL above. For LLNL, N-DIII (Arg1137–Ala1443) was amplified from full-length CaV2.2 and ligated into the AgeI/NotI sites of the engineered L-channel, thereby replacing L-DIII (Arg920–Ala1226). For LLLN, N-DIV (Pro1445–Ile1741) was amplified from full-length CaV2.2 and ligated into the NotI/BstEII sites of the engineered L-channel, thereby replacing L-DIV (Pro1228–Ile1542). For LNNL, N-DII and N-DIII were amplified from CaV2.2 with a 5′ primer containing an AscI site and a 3′ primer containing a NotI site, excised with AscI/NotI, and subcloned into the AscI/Not sites of LLNL. For LNNN, N-DIV was excised with NotI/XbaI from LLLN and subcloned into the NotI/XbaI sites of LNNL. For NNNL, N-DI was excised with HindIII/AscI from NLLL and subcloned into the HindIII/AscI sites of LNNL. For NNLN, N-DIV was excised with NotI/XbaI from LLLN and subcloned into the NotI/XbaI sites of NNLL. For NLLN, N-DI was excised with HindIII/AscI from NLLL and subcloned into the HindIII/AscI sites of LLLN. For L(l/n)LL, L-DII transmembrane segments 1–4 were amplified with a 5′ primer containing an AscI site and a 3′ primer (5′-ACC AGG TTC CTC AGG GAG TTC CAG TAC CTT GTA ATT TTG-3′) using NLLL as template, and N-DII transmembrane segments 5 and 6 with N-type intracellular linker between transmembrane segments 4 and 5 were amplified with a 5′ primer (5′-ATT ACA AGG TAC TGG AAC TCC CTG AGG AAC CTG GTT G-3′) and a 3′ primer lying downstream of an existing BfrI site in the WT α1C plasmid (pCDNA3). The final overlap PCR products were amplified using the above 5′ primer containing an AscI site and the above 3′ primer lying downstream of an existing BfrI site. The resulting PCR products were excised with AscI/BfrI and subcloned into the AscI/BfrI sites of LNLL to make L(l/n)LL. For LN*LL (also known as L(n/l)LL), N-DII transmembrane segments 1–4 were amplified with a 5′ primer containing an AscI site and a 3′ primer (5′-CAG GTT GCT CAA GGA GTT CCA ATA CTT GGT GAC TTT GAA AAT CCT CAG-3′) using LNLL as template, and L-DII transmembrane segments 5 and 6 with an L-type intracellular linker between transmembrane segments 4 and 5 were amplified with a 5′ primer (5′-GTC ACC AAG TAT TGG AAC TCC TTG AGC AAC CTG GTG GCC-3′) and a 3′ primer lying downstream of an existing BfrI site in WT α1C plasmid (pCDNA3). The final overlap PCR products were amplified using the above 5′ primer containing an AscI site and the above 3′ primer lying downstream of an existing BfrI site. The resulting PCR products were excised with AscI/BfrI and subcloned into the AscI/BfrI sites of LNLL to make L(n/l)LL. For LN*NN (also known as L(n/l)NN), first L-DIV in LLNL was replaced by N-DIV from LLLN to make a different LLNN maintaining the engineered unique AgeI site between the II and III domains, NotI sites between the III and IV domains, and XbaI site just after domain IV. Then the chimera domain II n/l was excised with AscI/BfrI from L(n/l)LL and subcloned into the AscI/BfrI sites of LLNN to make L(n/l)NN. For LN*NL (also known as L(n/l)NL), the fragment containing the chimera domain II n/l and N-DIII was excised with AscI/NotI from L(n/l)NN and ligated into the AscI/NotI sites of LNNL to make L(n/l)NL. The mutation and integrity of the mutant cDNAs was confirmed by qualitative restriction map analysis and directional DNA sequence analysis of the entire subcloned region. Functional expression of the mutant cDNAs was confirmed by Western blot analysis and patch clamp electrophysiology. We utilized either the calcium phosphate precipitation method (19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar) or Lipofectamine 2000 (following the manufacturer's directions) to transfect HEK 293 cells with WT L-, N-, and chimeric channels, which provided highly reproducible expression 24–72 h after transfection. HEK293 cells were maintained in standard Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% antibiotic-antimycotic mixtures (regular medium) at 37 °C in a 5% CO2 incubator. HEK293 cells were transfected with cDNA plasmids using the following molar ratio: 1 α1 subunit (L-, N-, or chimeric channel):1 α2δ:1 β1b:1 TAG (to increase expression efficiency):0.2 green fluorescent protein (to visualize transfected cells). The transfected cells were split next day into 35-mm dishes that served as the recording chamber. Cells were voltage-clamped using the whole-cell configuration of the patch clamp technique. Pipettes were pulled from Schott 8250 glass (Garner Glass, Claremont, CA) on a Sutter P-97 puller (Sutter Instruments Co., Novato, CA). Currents were recorded using an Axopatch 200A amplifier (Molecular Devices, Sunnyvale, CA) and digitized with the ITC-18 data acquisition interface (Instrutech Corp., Port Washington, NY). Experiments were controlled by a Power Macintosh G3 computer (Apple Computer, Cupertino, CA) running S5 data acquisition software written by Dr. Stephen Ikeda (NIAAA, National Institutes of Health, Bethesda, MD). Leak current was subtracted online using a P/4 protocol. All recordings were carried out at room temperature, and the holding potential was −120 mV. Whole-cell currents were digitized depending on test step duration at 50 (25 ms), 10 (200 ms), and 4 (1000 ms) kHz after analog filtering at 1–10 kHz. Data were analyzed using IgorPro (WaveMetrics, Lake Oswego, OR) running on a Macintosh computer. Step currents were measured as the average of 1 ms at the end of the voltage step. Activation τ (τAct) was determined by fitting a single exponential function to the step current after a 0.3-ms delay (15Buraei Z. Schofield G. Elmslie K.S. Neuropharmacology. 2007; 52: 883-894Crossref PubMed Scopus (45) Google Scholar). Inactivation τ (τInact) was determined by fitting a single exponential function from peak step current to the end of the step. The effect of roscovitine on inactivation of L-type or chimeric calcium channels was measured by using either 200- or 1000-ms voltage steps. The magnitude of inactivation was measured from either the IEnd/IPeak ratio, where IEnd was measured at the end of the step and IPeak was peak current, or as the IPost/IPre ratio from a triple pulse protocol consisting of identical 25-ms pre- and postpulse steps (to elicit peak current) bracketing a 200-ms test pulse to voltages ranging from −120 to +80 mV. Group data were calculated as mean ± S.D. throughout the paper. A paired t test was used for within-cell comparisons. One-way analysis of variance with Tukey's HSD post hoc test was used to test for differences among three or more independent groups. The internal pipette solution contained 104 mm NMG-Cl, 14 mm creatine-PO4, 6 mm MgCl2, 10 mm NMG-HEPES, 5 mm Tris-ATP, 0.3 mm Tris-GTP, and 10 mm NMG-EGTA with osmolarity of 280 mosm and pH 7.3. The external recording solution contained 30 mm BaCl2, 100 mm NMG-Cl, 10 mm NMG-HEPES, osmolarity of 300 mosm, and pH 7.3. We used Ba2+ as the permeant ion to exclude Ca2+-dependent inactivation (25Peterson B.Z. DeMaria C.D. Adelman J.P. Yue D.T. Neuron. 1999; 22: 549-558Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 26Peterson B.Z. Lee J.S. Mulle J.G. Wang Y. de Leon M. Yue D.T. Biophys. J. 2000; 78: 1906-1920Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), since we have previously demonstrated that (R)-roscovitine enhances VDI but does not affect calcium-dependent inactivation (19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar). Both (R)- and (S)-roscovitine were prepared as a 50 mm stock solution in DMSO and stored at −30 °C. All external solutions contained the same DMSO concentration so that the roscovitine concentration was the sole variable when changing solutions. Test solutions were applied from a gravity-fed perfusion system with an exchange time of 1–2 s. All experiments utilized (R)-roscovitine from LC Laboratories (Woburn, MA) and (S)-roscovitine from Alexis Biochemicals (San Diego, CA). Dulbecco's modified Eagle's medium/F-12, Dulbecco's modified Eagle's medium, fetal bovine serum, 100× antibiotic/antimycotic, and Lipofectamine 2000 were from Invitrogen. Other chemicals were obtained from Sigma. We have shown that (R)-roscovitine significantly slows activation and enhances inactivation of L-type channels and hypothesized that a single extracellular binding site mediated both effects (19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar). We further investigated this idea by examining the effect of (S)-roscovitine on L-type channels, which was useful in differentiating multiple roscovitine binding sites on N-type channels (15Buraei Z. Schofield G. Elmslie K.S. Neuropharmacology. 2007; 52: 883-894Crossref PubMed Scopus (45) Google Scholar, 17Cho S. Meriney S.D. Eur. J. Neurosci. 2006; 23: 3200-3208Crossref PubMed Scopus (29) Google Scholar, 18Buraei Z. Elmslie K.S. J. Neurochem. 2008; 105: 1450-1461Crossref PubMed Scopus (16) Google Scholar). Using 25-ms voltage steps to +20 mV, we verified that 100 μm (R)-roscovitine slowed L-channel activation (15Buraei Z. Schofield G. Elmslie K.S. Neuropharmacology. 2007; 52: 883-894Crossref PubMed Scopus (45) Google Scholar, 19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar) (Fig. 2). However, the activation rate was not altered by the application of 100 μm (S)-roscovitine (Fig. 2A), which was estimated by fitting the +20 mV step current using a single exponential function to generate τAct. The average change in τAct induced by (S)-roscovitine was 8.6 ± 8.5% (±S.D., n = 7), whereas the (R)-roscovitine-induced increase of τAct was 148.6 ± 24.8% (n = 9, p < 0.001; Fig. 2C). Thus, the L-channel binding site involved with slowed activation shows stereo-selectivity for roscovitine. The inability of (S)-roscovitine to slow L-channel activation could be explained by low affinity of a single roscovitine binding site for the S enantiomer. If this were true, inactivation should also be insensitive to (S)-roscovitine. This hypothesis was tested using 1000-ms voltage steps to +30 mV to measure VDI (30 mm Ba2+). Fig. 3 shows typical L-current traces recorded from the same cell exposed to either 100 μm (S)- or (R)-roscovitine. The speed of inactivation (τInact) was determined from single exponential fits to the inactivating portion of the current (peak to end). Counter to our hypothesis, (S)-roscovitine increased inactivation to a degree similar to that of (R)-roscovitine (Fig. 3). (S)-Roscovitine decreased τInact from 591 ± 133 to 220 ± 11 ms (p < 0.05, n = 3), whereas τInact was decreased from 504 ± 84 to 216 ± 7 ms (p < 0.05, n = 3) by (R)-roscovitine (Fig. 3B). There was no significant difference in τInact between (S)-roscovitine and (R)-roscovitine. The L-channel binding site that mediates roscovitine-induced enhancement of inactivation does not show stereo-selectivity. The effect of (S)-roscovitine on the voltage dependence of inactivation was determined using a three-pulse protocol where the ratio of the postpulse/prepulse (25 ms, +30 mV) current (IPost/IPre) was used to monitor inactivation induced by 200-ms inactivating steps ranging in voltage from −120 to +80 mV. Under control conditions (30 mm Ba2+), the inactivation versus voltage relationship was U-shaped (Fig. 3C), which was more prominent than we observed previously using 10 mm Ba2+ (19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar). The reason for this difference is unknown, but as we did with our previous data, we fit the inactivation versus voltage data from −120 to +50 mV (peak inactivation) using a single Boltzmann equation (Fig. 3C). (S)-Roscovitine increased the magnitude of inactivation so that the inactivation versus voltage relationship became less U-shaped. Maximal inactivation from the Boltzmann fit increased from 0.19 ± 0.01 in control and 0.18 ± 0.02 for recovery to 0.66 ± 0.03 by (S)-roscovitine. Boltzmann fitting of the IPost/IPre versus voltage relationship showed a small (∼5 mV) (S)-roscovitine-induced right shift in half-inactivation voltage (V½) and a small (e-fold/4 mV) decrease in slope (Fig. 3C) that was also observed with (R)-roscovitine (see Fig. 6A). Maximal inactivation was increased from 0.23 in control to 0.67 in (R)-roscovitine, which is very similar to our observations with (S)-roscovitine. These results suggest that the L-channel site mediating enhanced inactivation does not differentiate between (R)- and (S)-roscovitine. The dose-response relationship of (R)-roscovitine showed similar EC50 values for both slowed activation and enhanced inactivation (20–30 μm), which supported a single binding site (19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar). However, the Hill coefficient was close to 1 (1.2) for slowed activation, whereas that for enhanced inactivation was >2 (2.3), which suggested separate binding sites for these two effects. The differential sensitivity of slowed activation for (R)- versus (S)-roscovitine supports the latter idea. We further investigated the enhanced inactivation by determining the EC50 for (S)-roscovitine. Inactivation was measured as the IEnd/IPeak ratio calculated from 200-ms steps to +25 mV. (S)-Roscovitine increased inactivation in a dose-dependent manner from 10 to 100 μm, but the response to 300 μm was reproducibly reduced relative to that of 100 μm. This effect was also observed in (R)-roscovitine (19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar). The dose-response data were fit (0–100 μm) using the Hill equation to yield EC50 = 41.1 ± 0.1 μm and a Hill coefficient of 4.47 ± 0.03 (Fig. 4). Both of these values are larger than that obtained from (R)-roscovitine (19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar). One thing that is clear from these data is that the binding site mediating enhanced inactivation shows a Hill coefficient of >2, suggesting positive cooperativity. One explanation for this cooperativity is that roscovitine selectively binds to inactivated channels to enhance VDI. We were interested in localizing the L-type channel structures that mediate both roscovitine-induced effects to further investigate the hypothesis of multiple roscovitine binding sites. We focused on a chimeric strategy that had been previously exploited to determine DHP binding sites on L-type channels (20Grabner M. Wang Z. Hering S. Striessnig J. Glossmann H. Neuron. 1996; 16: 207-218Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) but needed to establish that the (R)-roscovitine effect on L-channels was unique. We had previously demonstrated that (R)-roscovitine uniquely affected L-channel activation because N-channel activation was slowed only at voltages of <0 mV and L-channel activation was slowed at all voltages (14Buraei Z. Anghelescu M. Elmslie K.S. Biophys. J. 2005; 89: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 19Yarotskyy V. Elmslie K.S. Br. J. Pharmacol. 2007; 152: 386-395Crossref PubMed Scopus (39) Google Scholar). However, our recordings of native N-current (bullfrog sympathetic neurons) showed enhanced inactivation during voltage steps (peak current) (14Buraei Z. Anghelescu M. Elmslie K.S. Biophys. J. 2005; 89: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Surprisingly, our data from N-type channels expressed in HEK293 cells failed to reproduce those results (FIGURE 5, FIGURE 6). Inactivation during 200-ms steps to +30 mV was not enhanced (Fig. 5B), and the mean IPost/IPre ratio was not changed at any voltage by 100 μm (R)-roscovitine (Fig. 6B). Single Boltzmann equation fits from −120 mV to peak inactivation (+10 mV) yielded maximum inactivation of 0.48 ± 0.05 in control versus 0.47 ± 0.03 (±S.D., n = 6, not significant) in (R)-roscovitine. Thus, (R)-roscovitine does not enhance U-type inactivation of N-type calcium channels (21Goo Y.S. Lim W. Elmslie K.S. J. Neurophysiol. 2006; 96: 1075-1083Crossref PubMed Scopus (7) Google Scholar) expressed in HEK293 cells. These" @default.
• W2010481540 created "2016-06-24" @default.
• W2010481540 creator A5002894637 @default.
• W2010481540 creator A5002965365 @default.
• W2010481540 creator A5017109296 @default.
• W2010481540 creator A5017404634 @default.
• W2010481540 creator A5053189843 @default.
• W2010481540 creator A5065957388 @default.
• W2010481540 date "2010-01-01" @default.
• W2010481540 modified "2023-09-28" @default.
• W2010481540 title "Roscovitine Binds to Novel L-channel (CaV1.2) Sites That Separately Affect Activation and Inactivation" @default.
• W2010481540 cites W1486098902 @default.
• W2010481540 cites W1510194880 @default.
• W2010481540 cites W1559359156 @default.
• W2010481540 cites W1599506780 @default.
• W2010481540 cites W1965769778 @default.
• W2010481540 cites W1970680117 @default.
• W2010481540 cites W1983526026 @default.
• W2010481540 cites W1995732275 @default.
• W2010481540 cites W2002326274 @default.
• W2010481540 cites W2007346956 @default.
• W2010481540 cites W2008250931 @default.
• W2010481540 cites W2019028325 @default.
• W2010481540 cites W2023331462 @default.
• W2010481540 cites W2026373451 @default.
• W2010481540 cites W2030759806 @default.
• W2010481540 cites W2038257888 @default.
• W2010481540 cites W2039771541 @default.
• W2010481540 cites W2045552946 @default.
• W2010481540 cites W2048912162 @default.
• W2010481540 cites W2063996715 @default.
• W2010481540 cites W2066205689 @default.
• W2010481540 cites W2071045396 @default.
• W2010481540 cites W2075440368 @default.
• W2010481540 cites W2078971375 @default.
• W2010481540 cites W2080780583 @default.
• W2010481540 cites W2083433702 @default.
• W2010481540 cites W2087062865 @default.
• W2010481540 cites W2101072772 @default.
• W2010481540 cites W2113662093 @default.
• W2010481540 cites W2121912757 @default.
• W2010481540 cites W2123374731 @default.
• W2010481540 cites W2138697269 @default.
• W2010481540 cites W2138773108 @default.
• W2010481540 cites W2140112927 @default.
• W2010481540 cites W2148274682 @default.
• W2010481540 cites W2159138670 @default.
• W2010481540 cites W2165564780 @default.
• W2010481540 cites W2165957980 @default.
• W2010481540 cites W2166375411 @default.
• W2010481540 cites W4231379732 @default.
• W2010481540 doi "https://doi.org/10.1074/jbc.m109.076448" @default.
• W2010481540 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2804190" @default.
• W2010481540 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19887376" @default.
• W2010481540 hasPublicationYear "2010" @default.
• W2010481540 type Work @default.
• W2010481540 sameAs 2010481540 @default.
• W2010481540 citedByCount "31" @default.
• W2010481540 countsByYear W20104815402012 @default.
• W2010481540 countsByYear W20104815402013 @default.
• W2010481540 countsByYear W20104815402014 @default.
• W2010481540 countsByYear W20104815402016 @default.
• W2010481540 countsByYear W20104815402017 @default.
• W2010481540 countsByYear W20104815402019 @default.
• W2010481540 countsByYear W20104815402020 @default.
• W2010481540 countsByYear W20104815402021 @default.
• W2010481540 countsByYear W20104815402022 @default.
• W2010481540 countsByYear W20104815402023 @default.
• W2010481540 crossrefType "journal-article" @default.
• W2010481540 hasAuthorship W2010481540A5002894637 @default.
• W2010481540 hasAuthorship W2010481540A5002965365 @default.
• W2010481540 hasAuthorship W2010481540A5017109296 @default.
• W2010481540 hasAuthorship W2010481540A5017404634 @default.
• W2010481540 hasAuthorship W2010481540A5053189843 @default.
• W2010481540 hasAuthorship W2010481540A5065957388 @default.
• W2010481540 hasBestOaLocation W20104815402 @default.
• W2010481540 hasConcept C12554922 @default.
• W2010481540 hasConcept C15744967 @default.
• W2010481540 hasConcept C185592680 @default.
• W2010481540 hasConcept C2776035688 @default.
• W2010481540 hasConcept C46312422 @default.
• W2010481540 hasConcept C86803240 @default.
• W2010481540 hasConceptScore W2010481540C12554922 @default.
• W2010481540 hasConceptScore W2010481540C15744967 @default.
• W2010481540 hasConceptScore W2010481540C185592680 @default.
• W2010481540 hasConceptScore W2010481540C2776035688 @default.
• W2010481540 hasConceptScore W2010481540C46312422 @default.
• W2010481540 hasConceptScore W2010481540C86803240 @default.
• W2010481540 hasIssue "1" @default.
• W2010481540 hasLocation W20104815401 @default.
• W2010481540 hasLocation W20104815402 @default.
• W2010481540 hasLocation W20104815403 @default.
• W2010481540 hasLocation W20104815404 @default.
• W2010481540 hasOpenAccess W2010481540 @default.
• W2010481540 hasPrimaryLocation W20104815401 @default.
• W2010481540 hasRelatedWork W1531601525 @default.
• W2010481540 hasRelatedWork W1990781990 @default.
• W2010481540 hasRelatedWork W2319480705 @default.

• next