FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2029372013> ?p ?o ?g. }

• W2029372013 endingPage "32654" @default.
• W2029372013 startingPage "32647" @default.
• W2029372013 abstract "Voltage-gated sodium channels consist of a pore-forming α subunit associated with β1 subunits and, for brain sodium channels, β2 subunits. Although much is known about the structure and function of the α subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) α subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) α subunit. In comparison with rH1, wild-type rIIA α subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of β1 subunits inXenopus oocytes. When α subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA α subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA α subunits, all of the chimeric rIIA α subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with β1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of β1 subunits in shifting rIIA α subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by β1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation. Voltage-gated sodium channels consist of a pore-forming α subunit associated with β1 subunits and, for brain sodium channels, β2 subunits. Although much is known about the structure and function of the α subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) α subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) α subunit. In comparison with rH1, wild-type rIIA α subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of β1 subunits inXenopus oocytes. When α subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA α subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA α subunits, all of the chimeric rIIA α subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with β1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of β1 subunits in shifting rIIA α subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by β1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation. rat IIA rat H1 wild-type Voltage-gated sodium channels mediate the sodium conductance responsible for the rapidly rising phase of the action potential in nerve and muscle cells. The major form of the sodium channel in rat brain is a heterotrimeric complex of an α subunit (260 kDa), a noncovalently bound β1 subunit (36 kDa), and a disulfide-linked β2 subunit (33 kDa) (1Catterall W.A. Science. 1984; 223: 653-661Crossref PubMed Scopus (233) Google Scholar, 2Catterall W.A. Physiol. Rev. 1992; 72: S15-S48Crossref PubMed Google Scholar). α subunits can function as voltage-gated ion channels by themselves (e.g. rat brain type II/IIA; Refs. 3Goldin A.L. Snutch T. Lubbert H. Dowsett A. Marshall J. Auld V. Downey W. Fritz L.C. Lester H.A. Dunn R. Catterall W.A. Davidson N. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7503-7507Crossref PubMed Scopus (149) Google Scholar and 4Noda M. Ikeda T. Suzuki T. Takeshima H. Takahashi T. Kuno M. Numa S. Nature. 1986; 322: 826-828Crossref PubMed Scopus (393) Google Scholar). They are composed of four homologous domains (I–IV), which each contain six probable α-helical transmembrane segments (S1–S6) and an additional membrane-associated pore loop (e.g. rat brain type II/IIA; Refs. 5Noda M. Ikeda T. Kayano T. Suzuki H. Takeshima H. Kurasaki M. Takahashi H. Numa S. Nature. 1986; 320: 188-192Crossref PubMed Scopus (686) Google Scholar, 6Auld V.J. Goldin A.L. Krafte D.S. Marshall J. Dunn J.M. Catterall W.A. Lester H.A. Davidson N. Dunn R.J. Neuron. 1988; 1: 449-461Abstract Full Text PDF PubMed Scopus (298) Google Scholar, 7Auld V.J. Goldin A.L. Krafte D.S. Catterall W.A. Lester H.A. Davidson N. Dunn R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 323-327Crossref PubMed Scopus (163) Google Scholar), whereas the β1 and β2 subunits are single membrane-spanning glycoproteins with a large extracellular domain and a small intracellular domain (8Isom L.L. De Jongh K.S. Patton D.E. Reber B.F.X. Offord J. Charbonneau H. Walsh K. Goldin A.L. Catterall W.A. Science. 1992; 256: 839-842Crossref PubMed Scopus (598) Google Scholar, 9Isom L.L. Ragsdale D.S. De Jongh K.S. Westenbroek R.E. Reber B.F.X. Scheuer T. Catterall W.A. Cell. 1995; 83: 433-442Abstract Full Text PDF PubMed Scopus (395) Google Scholar). Extensive structure-function analyses of α subunits have shown that the S4 transmembrane segments in each domain serve as voltage sensors for channel activation; the S5 and S6 segments and the pore loop between them form the transmembrane pore; and the short, highly conserved intracellular loop between domains III and IV forms the inactivation gate (reviewed in Refs. 10Catterall W.A. Annu. Rev. Biochem. 1995; 65: 493-531Crossref Scopus (762) Google Scholar and 11Fozzard H.A. Hanck D.A. Physiol. Rev. 1996; 76: 887-926Crossref PubMed Scopus (233) Google Scholar). The large intracellular domains are targets for channel modulation by protein phosphorylation and G protein binding (reviewed in Ref. 12Catterall W.A. Adv. Second Messenger Phosphoprotein Res. 1997; 31: 159-181Crossref PubMed Google Scholar). In contrast to the well established functional roles of the transmembrane and intracellular domains of the channel, the functional roles of the extracellular loops of the sodium channel α subunit have not been defined. Peptide neurotoxins from scorpions and sea anemones modulate gating by binding to receptor sites in the extracellular domains (13Rogers J.C. Qu Y. Tanada T.N. Scheuer T. Catterall W.A. J. Biol. Chem. 1996; 271: 15950-15962Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 14Cestèle S. Qu Y. Rogers J.C. Rochat H. Scheuer T. Catterall W.A. Neuron. 1998; 21: 919-931Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar), and the extracellular domain of the β1 subunit is primarily responsible for its modulation of α subunit function (15Chen C.F. Cannon S.C. Pfluegers Arch. Eur. J. Physiol. 1995; 431: 186-195Crossref PubMed Scopus (67) Google Scholar, 16Makita N. Bennett Jr., P.B. George Jr., A.L. J. Neurosci. 1996; 16: 7117-7127Crossref PubMed Google Scholar, 17McCormick K.A. Isom L.L. Ragsdale D. Smith D. Scheuer T. Catterall W.A. J. Biol. Chem. 1998; 273: 3954-3962Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These results suggest that the extracellular loops of α subunits might also be important determinants of sodium channel gating. Cardiac sodium channel α subunits (type H1) (18Rogart R.B. Cribbs L.L. Muglia L.K. Kephart D.D. Kaiser M.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8170-8174Crossref PubMed Scopus (307) Google Scholar, 19Kallen R.G. Sheng Z.H. Yang J. Chen L.-Q. Rogart R.B. Barchi R.L. Neuron. 1990; 4: 233-242Abstract Full Text PDF PubMed Scopus (229) Google Scholar) differ substantially from brain type IIA in their voltage dependence and kinetics of activation and inactivation and in their response to association with β1 subunits (11Fozzard H.A. Hanck D.A. Physiol. Rev. 1996; 76: 887-926Crossref PubMed Scopus (233) Google Scholar). In this study, we have analyzed the functional properties and the modulation by the β1 subunit of chimeras constructed between rat brain type IIA (rIIA)1 and rat heart (rH1) sodium channel α subunits to reveal functional activities of the extracellular loops of the α subunit and to identify specific extracellular segments that are important determinants of voltage-dependent gating and interaction with the β1 subunit. Five M13 constructs collectively spanning nearly the entire rIIA sequence were used as templates for site-directed mutagenesis. The template mp18SSNC (nucleotides 1898–2700) contains the SmaI/SphI rIIA fragment. The template mp19BstNC was created by first introducing a BstEII site in the mp19 vector and then subcloning theSphI/BstEII rIIA fragment (nucleotides 2701–4636). The remaining three templates, mp18KXNC (nucleotides 23–540), mp19XaI (nucleotides 541–1897), and mp18RVNC (nucleotides 4279–5997) have been described (13Rogers J.C. Qu Y. Tanada T.N. Scheuer T. Catterall W.A. J. Biol. Chem. 1996; 271: 15950-15962Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Of the 16 extracellular loops, one loop is identical between the rIIA and rH1 isoforms (IS3-S4), and 12 of the remaining loops were short enough to use oligonucleotide-directed mutagenesis to directly introduce the rIIA-to-rH1 amino acid changes, using uracil-containing templates and the dut−ung− selection procedure (20Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4880) Google Scholar). The three remaining large loop chimeras (IS5-SS1, IIIS5-SS1, and IVSS2-S6) were created by using oligonucleotide-directed mutagenesis to delete the sequence encoding each loop while simultaneously introducing silent restriction sites flanking these individual regions. Cardiac-specific primers containing the flanking restriction sites were then used to amplify the sequence encoding each corresponding loop from rH1 cDNA for cloning into the appropriate region of rIIA, as described previously (13Rogers J.C. Qu Y. Tanada T.N. Scheuer T. Catterall W.A. J. Biol. Chem. 1996; 271: 15950-15962Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Fragments containing mutations were then excised from these mutagenesis templates and cloned into pCDM8SalK-NC or pCDM8Sal-NC. All mutations were confirmed in the final constructs by DNA sequencing and extensive restriction mapping. pCDM8 plasmids encoding WT and chimeric sodium channel α subunits were linearized withClaI, and plasmids encoding β1 subunits were linearized with HindIII. Transcription was performed with T7 RNA polymerase (Ambion Inc., Austin, TX). Isolation, preparation, and maintenance of Xenopus oocytes were carried out as described previously (21McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 12025-12034Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Healthy oocytes selected manually were pressure-injected with 50 nl of a solution containing either a 1:4 or 1:1 molar ratio of α to β1 subunit RNA. Electrophysiological recordings were carried out 2–5 days after injection. Two-electrode voltage-clamp experiments were performed as described previously (22Qu Y. Isom L.L. Westenbroek R.E. Rogers J.C. Tanada T.N. McCormick K.A. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 25696-25701Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The amplitude of expressed sodium currents was typically 0.5–5 μA. The voltage-clamp protocols are described in the figure legends. Conductance-voltage (g-V) relationships were derived from current-voltage (I-V) relationships according to g = I/(V −V r ), where I is the peak current amplitude measured at voltage V and the reversal potentialV r is assumed to be +55 mV under our recording conditions. Normalized g-V relationships and inactivation curves were fit with a Boltzmann distribution, 1/(1 + exp((V − V 12)/k)), where V 12 is the voltage at which half-activation or half-inactivation occurred and k is a slope factor. The time courses of current decay and recovery from inactivation were described with two exponentials, a(1 − exp(−t/τ1)) + (1 − a)(1 − exp(−t/τ2)), where a is the fraction of the fast component, τ1 is the time constant of the fast component, and τ2 is the time constant of the slow component. Pooled data are reported as means ± S.D. Statistical comparisons were done using Student's t test, with p < 0.05 as the criterion for significance. Compared with rat brain sodium channels, rat cardiac sodium channels containing the rH1 α subunit (18Rogart R.B. Cribbs L.L. Muglia L.K. Kephart D.D. Kaiser M.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8170-8174Crossref PubMed Scopus (307) Google Scholar, 19Kallen R.G. Sheng Z.H. Yang J. Chen L.-Q. Rogart R.B. Barchi R.L. Neuron. 1990; 4: 233-242Abstract Full Text PDF PubMed Scopus (229) Google Scholar) activate and inactivate at more negative membrane potentials, and the kinetics of inactivation and recovery from inactivation are faster when expressed in Xenopus oocytes (11Fozzard H.A. Hanck D.A. Physiol. Rev. 1996; 76: 887-926Crossref PubMed Scopus (233) Google Scholar, 23Cribbs L.L. Satin J. Fozzard H.A. Rogart R.B. FEBS Lett. 1990; 275: 195-200Crossref PubMed Scopus (52) Google Scholar, 24White M.M. Chen L.-Q. Kleinfield R. Kallen R.G. Barchi R.L. Mol. Pharmacol. 1991; 39: 604-608PubMed Google Scholar). The electrophysiological properties of the two wild-type sodium channels and each chimeric channel were analyzed in two-microelectrode whole-cell voltage-clamp experiments inXenopus oocytes. Fig. 1 shows typical sodium currents elicited from WT rIIA and rH1 α subunits expressed alone in Xenopus oocytes. The resolution of the two-microelectrode voltage clamp of small oocytes (25Krafte D.S. Lester H.A. J. Neurosci. Methods. 1989; 26: 211-215Crossref PubMed Scopus (12) Google Scholar) was adequate for detailed analysis of the kinetics of inactivation, but the rate of activation was not resolved with sufficient precision for accurate analysis. As reported previously (23Cribbs L.L. Satin J. Fozzard H.A. Rogart R.B. FEBS Lett. 1990; 275: 195-200Crossref PubMed Scopus (52) Google Scholar, 24White M.M. Chen L.-Q. Kleinfield R. Kallen R.G. Barchi R.L. Mol. Pharmacol. 1991; 39: 604-608PubMed Google Scholar), the rate of inactivation of the rH1 α subunit was faster than that of the rIIA α subunit expressed alone in Xenopus oocytes (Fig. 1, A andB). Coexpression of β1 subunits greatly accelerated inactivation of rIIA α subunits (Fig. 1 A), but had little effect on the rH1 α subunit (Fig. 1 B). Modulation by the β1 subunits therefore results in faster activation and inactivation of wild-type rIIA than rH1 α subunits. To examine the functional role of the extracellular loops in the sodium channel α subunit, we substituted each predicted extracellular loop in the brain rIIA α subunit individually with the corresponding sequence of the heart sodium channel rH1 α subunit (TableI), except for the loop between S3 and S4 in domain I, which is identical between rIIA and rH1. No sodium current was detected when oocytes were injected with mRNAs from two chimeras, one having substitutions in loop IIS1-S2 and the other in IIS3-S4. Although these two extracellular loop chimeras were not analyzed in this study, a set of single amino acid chimeras containing each of the amino acid differences in these two extracellular loops was analyzed previously, and no changes in sodium channel gating or response to β1 subunits were observed (14Cestèle S. Qu Y. Rogers J.C. Rochat H. Scheuer T. Catterall W.A. Neuron. 1998; 21: 919-931Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). 2Y. Qu, J. C. Rogers, S.-F. Chen, K. A. McCormick, T. Scheuer, and W. A. Catterall, unpublished results. Each of the other 13 extracellular loop chimeras gave sodium currents sufficient for detailed analysis (0.5–5 μA). For most chimeras, the expression level was comparable to that of WT when 20 ng of each mRNA was injected. For chimeras with lower expression levels than WT, up to 160 ng of α subunit mRNA was injected to obtain sodium currents of similar amplitude to those observed for 20 ng of WT α subunit mRNA.Table IAmino acid sequences of sodium channel chimerasExtracellular loopSequenceIS1-S2 rIIaSNPPDWTKN ChangesHD––P–––YIS3-S4 rIIaFVDLGNVSAL Changes––––––––––IS5-SS1 rIIaNKCLQWPPDN STFEINITSF FNNSLDWNGT AFNRTVNMFN ChangesH––VR––––– NFT–L–G––– ––G–VEAD–L VW––––––NS rIIaWDEYIEDKSH FYFLEGQNDA LLCGNSSDAG QCPEGYICVK AGRNPNYGYT SF ChangesL–V–LN–PAN YLLKN–TT–V –––––––––– T–––––R–L– ––E––DH––– ––ISS2-S6 rIIaNLYQLTLRAA GKTY ChangesR–––Q–––S– ––I–IIS1-S2 rIIaEHYPMTEQFS SV Changes–––N––AE–E EMIIS3-S4 rIIaANVEGLS ChangesSRMGN––IIS5-SS1 rIIaKSYKECVCKI SNDCELPRWH MHH Changes–N–S–LRHR– –DSGL––––– –MDIISS2-S6 rIIaETMWDCMEVA GQT Changes–––––––––S ––SIIIS1-S2 rIIaEDIYIEQRKT IKT Changes––––L–E––– ––VIIIS3-S4 rIIaALGYSE Changes–T––YAIIIS5-SS1 rIIaKFYHCINYTT GEM.FDVSVV NNYSECQALI ESNQTARWKN VKVNFD Changes––GR–––Q–E –DLPLNYTI– ––K–––ESFN VTGELYWT–– ––––––IIISS2-S6 rIIaDIMYAAVDSR NVELQPKYED Changes–––––––––– GY–E––QW––IVS1-S2 rIIaETDDQSQEMT N Changes––––––P–KV –IVS3-S4 rIIaKYFVSPTLF Changes–––F–––––IVS5-SS1 rIIaREVGIDDMFN FE ChangesW–A––––––– –QIVSS2-S6 rIIaGLLAPILNSG PPDCDPEKDH PGSSVKGDCG NPS Changes–––S––––T– ––Y–––NLPN SNG––R–N–– S–A Open table in a new tab The voltage dependence of activation of WT rIIA and rH1 α subunits and selected α subunit chimeras expressed alone in Xenopus oocytes is illustrated as conductance-voltage curves in Fig.2 A, and the mean values forV a, the voltage for half-maximal activation, are presented in Fig. 2 C. V a for rH1 is 18 mV more negative than for rIIA (Fig. 2, A and C,open symbols). Five chimeras (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had a more negative voltage dependence of activation than WT rIIA (Fig. 2, A and C). Only chimera IVSS2-S6 was observed to have a slightly more positive voltage dependence of activation than WT rIIA. Thus, these extracellular loops are important determinants of the voltage dependence of activation and contribute to the more negative voltage dependence observed for cardiac rH1 channels. If their effects were additive, they would more than account for the difference in activation gating between the two channels. The voltage dependence of steady-state inactivation was studied with 100-ms conditioning prepulses. With this protocol, the voltage for half-inactivation of rH1 was 17 mV more negative than that of rIIA (Fig. 2 B). Three chimeras (IISS2-S6, IIIS1-S2, and IVS3-S4) inactivated at more negative potentials than WT rIIA, but at more positive potentials than rH1 (Fig. 2 B). These were the only chimeras that had a shifted voltage dependence of inactivation (Fig.2 D). The three chimeras whose voltage dependence of inactivation was negatively shifted (IISS2-S6, IIIS1-S2, and IVS3-S4) also had a negatively shifted voltage dependence of activation (Fig. 2,C and D), suggesting that the change in inactivation results from coupling to the negatively shifted activation. Thus, these three extracellular loops are important determinants of the voltage dependence of inactivation as well as activation. Together, the negative shifts in steady-state inactivation observed for these three chimeras would be more than sufficient to account for the difference in the voltage dependence of inactivation between the rIIA and rH1 α subunits. Expression of the WT rIIA α subunit alone inXenopus oocytes produced a slowly inactivating sodium current (Fig. 3 A, slow solid lines). The time courses of the decay of these sodium currents are well fit by a sum of fast and slow exponentials, with the fraction of fast inactivation (F fast) less than 0.1 for rIIA α subunits expressed alone and near 1.0 for rH1 expressed alone (Fig. 3 E). The slowly inactivating component of this biphasic time course has previously been shown to result from a slow gating mode that is particularly prominent for type IIA α subunits expressed alone in Xenopus oocytes (7Auld V.J. Goldin A.L. Krafte D.S. Catterall W.A. Lester H.A. Davidson N. Dunn R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 323-327Crossref PubMed Scopus (163) Google Scholar, 26Moorman J.R. Kirsch G.E. VanDongen A.M. Joho R.H. Brown A.M. Neuron. 1990; 4: 243-252Abstract Full Text PDF PubMed Scopus (116) Google Scholar, 27Zhou J. Potts J.F. Trimmer J.S. Agnew W.S. Sigworth F.J. Neuron. 1991; 7: 755-785Abstract Full Text PDF PubMed Scopus (122) Google Scholar). It is not thought to be related to the distinct slow inactivation process of sodium channels, which requires longer depolarization and involves a different gating mechanism than fast inactivation (28Adelman W.J. Palti Y. J. Gen. Physiol. 1968; 54: 589-606Crossref Scopus (113) Google Scholar, 29Narahashi T. J. Cell. Comp. Physiol. 1964; 64: 73-96Crossref PubMed Google Scholar, 30Ogata N. Tatebayashi H. J. Membr. Biol. 1992; 129: 71-80Crossref PubMed Scopus (36) Google Scholar, 31Rudy B. J. Physiol. (Lond.). 1978; 283: 1-21Crossref Scopus (208) Google Scholar, 32Featherstone D.E. Richmond J.E. Ruben P.C. Biophys. J. 1996; 71: 3098-3109Abstract Full Text PDF PubMed Scopus (109) Google Scholar). Most chimeras displayed a similar time course of inactivation to that of WT at a test potential of +10 mV. However, five chimeras (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) inactivated faster than WT (Fig. 3, A–D). As for WT, analysis of these sodium currents revealed that they could be fit by two exponential functions with fixed time constants, but with an increased fraction of channels inactivating with the faster time constant (Fig. 3 E). These results indicate that the differences of these chimeric channels from WT are caused by a shift of the chimeric channels from the slow to the fast gating mode. Thus, these five extracellular segments are important determinants of the channel gating mode. The rate of recovery from inactivation at negative holding potentials was studied by repolarization to −100 mV following a conditioning pulse to −10 mV for 300 ms. As for inactivation, the time course of recovery from inactivation for WT rIIA channels expressed without β1 subunits can be described by a sum of two exponentials, with approximately half of the channels recovering rapidly from inactivation (Fig. 4 A). Using this protocol, the fraction of fast recovery from inactivation is ∼0.6 for rIIA and 0.9 for rH1 (Fig. 4 E). For most chimeric channels, the fraction of fast recovery from inactivation was similar to that of WT rIIA α subunits (Fig. 4 E). However, four chimeras that inactivated rapidly (IS5-SS1, ISS2-S6, IISS2-S6, and IIIS1-S2) also had faster kinetics of recovery from inactivation compared with WT rIIA channels (Fig. 4, A–D, closed circles for chimeras versus open circles for rIIA). This accelerated recovery from inactivation was caused by a shift of channels from the slow gating mode to the fast gating mode, as illustrated in the measurements of F fast in Fig.4 E. The effects of these chimeric substitutions on the kinetics of inactivation and recovery from inactivation indicate that four extracellular loops in domains I and III are important determinants of the sodium channel gating mode and that segment IVS3-S4 also has a minor effect. These are the first regions of the sodium channel structure found to have specific effects on the gating mode. As for the effects on voltage dependence, these effects of chimeric substitutions on the gating mode would be sufficient to fully account for the difference between rIIA and rH1 if they were additive. When either brain or skeletal muscle sodium channel α subunits are expressed alone in Xenopus oocytes, the resulting sodium currents are small, and they activate and inactivate abnormally slowly (6Auld V.J. Goldin A.L. Krafte D.S. Marshall J. Dunn J.M. Catterall W.A. Lester H.A. Davidson N. Dunn R.J. Neuron. 1988; 1: 449-461Abstract Full Text PDF PubMed Scopus (298) Google Scholar, 26Moorman J.R. Kirsch G.E. VanDongen A.M. Joho R.H. Brown A.M. Neuron. 1990; 4: 243-252Abstract Full Text PDF PubMed Scopus (116) Google Scholar, 27Zhou J. Potts J.F. Trimmer J.S. Agnew W.S. Sigworth F.J. Neuron. 1991; 7: 755-785Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 33Ji S. Sun W. George Jr., A.L. Horn R. Barchi R.L. J. Gen. Physiol. 1994; 104: 625-643Crossref PubMed Scopus (42) Google Scholar). Coexpression of the β1 subunit increases the current amplitude, negatively shifts the voltage dependence of activation and inactivation, and accelerates the rate of inactivation and recovery from inactivation (8Isom L.L. De Jongh K.S. Patton D.E. Reber B.F.X. Offord J. Charbonneau H. Walsh K. Goldin A.L. Catterall W.A. Science. 1992; 256: 839-842Crossref PubMed Scopus (598) Google Scholar, 34Makita N. Bennett Jr., P.B. George Jr., A.L. J. Biol. Chem. 1994; 269: 7571-7578Abstract Full Text PDF PubMed Google Scholar, 35Patton D.E. Isom L.L. Catterall W.A. Goldin A.L. J. Biol. Chem. 1994; 269: 17649-17655Abstract Full Text PDF PubMed Google Scholar, 36Cannon S.C. McClatchey A.I. Gusella J.F. Pfluegers Arch. Eur. J. Physiol. 1993; 423: 155-157Crossref PubMed Scopus (79) Google Scholar, 37Bennett Jr., P.B. Makita N. George Jr., A.L. FEBS Lett. 1993; 326: 21-24Crossref PubMed Scopus (73) Google Scholar, 38Wallner M. Weigl L. Meera P. Lotan I. FEBS Lett. 1993; 336: 535-539Crossref PubMed Scopus (49) Google Scholar, 39Yang J.S. Bennett P.B. Makita N. George A.L. Barchi R.L. Neuron. 1993; 11: 915-922Abstract Full Text PDF PubMed Scopus (60) Google Scholar), as illustrated for type IIA sodium channels in Figs. 1 A, 3 A, and4 A. The acceleration of inactivation and recovery from inactivation are thought to result from a shift of the rIIA α subunit from a slow gating mode to a fast gating mode upon coexpression of β1 subunits (35Patton D.E. Isom L.L. Catterall W.A. Goldin A.L. J. Biol. Chem. 1994; 269: 17649-17655Abstract Full Text PDF PubMed Google Scholar, 37Bennett Jr., P.B. Makita N. George Jr., A.L. FEBS Lett. 1993; 326: 21-24Crossref PubMed Scopus (73) Google Scholar, 38Wallner M. Weigl L. Meera P. Lotan I. FEBS Lett. 1993; 336: 535-539Crossref PubMed Scopus (49) Google Scholar, 40Chang S.Y. Satin J. Fozzard H.A. Biophys. J. 1996; 70: 2581-2592Abstract Full Text PDF PubMed Scopus (30) Google Scholar). β1 subunit mRNA is expressed in cardiac myocytes, as assessed by high resolution in situhybridization (22Qu Y. Isom L.L. Westenbroek R.E. Rogers J.C. Tanada T.N. McCormick K.A. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 25696-25701Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Coexpression of the rat β1 subunit with the rH1 α subunit significantly increases the current amplitude inXenopus oocytes, but the voltage dependence and kinetics of gating are not dramatically altered (22Qu Y. Isom L.L. Westenbroek R.E. Rogers J.C. Tanada T.N. McCormick K.A. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 25696-25701Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Similar experiments with human cardiac α and β1 subunits revealed significant effects of coexpression of β1 subunits on the voltage dependence of inactivation, but these effects were much smaller than those observed for brain or skeletal muscle sodium channels (41Makielski J.C. Limberis J.T. Chang S.Y. Fan Z. Kyle J.N. Mol. Pharmacol. 1996; 49: 30-39PubMed Google Scholar, 42Nuss H.B. Chiamvimonvat N. Pérez-Garcia M.T. Tomaselli G.F. Marbán E. J. Gen. Physiol. 1995; 106: 1171-1191Crossref PubMed Scopus (120) Google Scholar). These differences in response to coexpression of β1 subunits suggested that analysis of brain/cardiac sodium channel chimeras may reveal extracellular loops required for β1 subunit binding or modulation of α subunit function. Effects of β1 subunits on the sodium channel gating mode are most easily assessed from measurement of the kinetics of inactivation and recovery from inactivation and analysis by exponential curve fitting. Coexpression of the β1 subunit substantially accelerated the kinetics of inactivation of rIIA (Fig.3 A, fast solid lines). The increased rate of inactivation was fit by an increase in F fastfrom <0.1 to ∼0.9 for an α/β1 subunit RNA ratio of 1:4 (Fig. 3,E and F). Coexpression of β1 subunits at that level substantially accelerated the rate of inactivation of all chimeras (Fig. 3, A–D, dotted lines). With an α/β1 subunit RNA molar ratio of 1:4, most chimeric channels displayed a fraction of fast inactivation near 0.9, comparable to WT rIIA in the presence of β1 subunits (Fig. 3 F). In contrast, chimera IVSS2-S6 showed a substantially decreased fraction of fast inactivating channels compared with WT in the presence of β1 subunits (Fig. 3 F). Since its voltage dependence of inactivation was not significantly different" @default.
• W2029372013 created "2016-06-24" @default.
• W2029372013 creator A5002307585 @default.
• W2029372013 creator A5003453767 @default.
• W2029372013 creator A5028040253 @default.
• W2029372013 creator A5075205709 @default.
• W2029372013 creator A5077986587 @default.
• W2029372013 creator A5078941732 @default.
• W2029372013 date "1999-11-01" @default.
• W2029372013 modified "2023-09-27" @default.
• W2029372013 title "Functional Roles of the Extracellular Segments of the Sodium Channel α Subunit in Voltage-dependent Gating and Modulation by β1 Subunits" @default.
• W2029372013 cites W1509097602 @default.
• W2029372013 cites W1836738800 @default.
• W2029372013 cites W1965522673 @default.
• W2029372013 cites W1966093407 @default.
• W2029372013 cites W1967204209 @default.
• W2029372013 cites W1968903551 @default.
• W2029372013 cites W1974794546 @default.
• W2029372013 cites W1975793339 @default.
• W2029372013 cites W1977073662 @default.
• W2029372013 cites W1980378882 @default.
• W2029372013 cites W1986792633 @default.
• W2029372013 cites W1988669735 @default.
• W2029372013 cites W1997935314 @default.
• W2029372013 cites W2001539229 @default.
• W2029372013 cites W2003360892 @default.
• W2029372013 cites W2004002713 @default.
• W2029372013 cites W2006271002 @default.
• W2029372013 cites W2011889469 @default.
• W2029372013 cites W2012106712 @default.
• W2029372013 cites W2035565538 @default.
• W2029372013 cites W2036931150 @default.
• W2029372013 cites W2038702386 @default.
• W2029372013 cites W2044362364 @default.
• W2029372013 cites W2055170574 @default.
• W2029372013 cites W2056469720 @default.
• W2029372013 cites W2065639286 @default.
• W2029372013 cites W2065928015 @default.
• W2029372013 cites W2066707647 @default.
• W2029372013 cites W2067576287 @default.
• W2029372013 cites W2073929496 @default.
• W2029372013 cites W2086956125 @default.
• W2029372013 cites W2092435801 @default.
• W2029372013 cites W2094362927 @default.
• W2029372013 cites W2103375731 @default.
• W2029372013 cites W2104836764 @default.
• W2029372013 cites W2112571943 @default.
• W2029372013 cites W2114457659 @default.
• W2029372013 cites W2118426007 @default.
• W2029372013 cites W2125499229 @default.
• W2029372013 cites W2152715004 @default.
• W2029372013 cites W2163279702 @default.
• W2029372013 cites W2165071497 @default.
• W2029372013 cites W2165605060 @default.
• W2029372013 cites W2171411680 @default.
• W2029372013 cites W2171744066 @default.
• W2029372013 cites W2172530119 @default.
• W2029372013 cites W2228644631 @default.
• W2029372013 cites W2255288257 @default.
• W2029372013 cites W2460864356 @default.
• W2029372013 cites W4244153167 @default.
• W2029372013 doi "https://doi.org/10.1074/jbc.274.46.32647" @default.
• W2029372013 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10551819" @default.
• W2029372013 hasPublicationYear "1999" @default.
• W2029372013 type Work @default.
• W2029372013 sameAs 2029372013 @default.
• W2029372013 citedByCount "73" @default.
• W2029372013 countsByYear W20293720132012 @default.
• W2029372013 countsByYear W20293720132013 @default.
• W2029372013 countsByYear W20293720132014 @default.
• W2029372013 countsByYear W20293720132015 @default.
• W2029372013 countsByYear W20293720132016 @default.
• W2029372013 countsByYear W20293720132017 @default.
• W2029372013 countsByYear W20293720132018 @default.
• W2029372013 countsByYear W20293720132019 @default.
• W2029372013 countsByYear W20293720132022 @default.
• W2029372013 countsByYear W20293720132023 @default.
• W2029372013 crossrefType "journal-article" @default.
• W2029372013 hasAuthorship W2029372013A5002307585 @default.
• W2029372013 hasAuthorship W2029372013A5003453767 @default.
• W2029372013 hasAuthorship W2029372013A5028040253 @default.
• W2029372013 hasAuthorship W2029372013A5075205709 @default.
• W2029372013 hasAuthorship W2029372013A5077986587 @default.
• W2029372013 hasAuthorship W2029372013A5078941732 @default.
• W2029372013 hasBestOaLocation W20293720131 @default.
• W2029372013 hasConcept C104292427 @default.
• W2029372013 hasConcept C104317684 @default.
• W2029372013 hasConcept C121332964 @default.
• W2029372013 hasConcept C123079801 @default.
• W2029372013 hasConcept C12554922 @default.
• W2029372013 hasConcept C127162648 @default.
• W2029372013 hasConcept C178790620 @default.
• W2029372013 hasConcept C185592680 @default.
• W2029372013 hasConcept C194544171 @default.
• W2029372013 hasConcept C24890656 @default.
• W2029372013 hasConcept C28406088 @default.
• W2029372013 hasConcept C41008148 @default.
• W2029372013 hasConcept C50952357 @default.

• next