Structural Determinants of the Regulation of the Voltage-gated Potassium Channel Kv2.1 by the Modulatory α-Subunit Kv9.3
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m213117200
ISSN1083-351X
AutoresDaniel Kerschensteiner, Francisco J. Monje, Martin Stocker,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoVoltage-gated potassium (Kv) channels containing α-subunits of the Kv2 subfamily mediate delayed rectifier currents in excitable cells. Channels formed by Kv2.1 α-subunits inactivate from open- and closed states with both forms of inactivation serving different physiological functions. Here we show that open- and closed-state inactivation of Kv2.1 can be distinguished by the sensitivity to intracellular tetraethylammonium and extracellular potassium and lead to the same inactivated conformation. The functional properties of Kv2.1 are regulated by its association with modulatory α-subunits (Kv5, Kv6, Kv8, and Kv9). For instance, Kv9.3 changes the state preference of Kv2.1 inactivation by accelerating closed-state inactivation and inhibiting open-state inactivation. An N-terminal regulatory domain (NRD) has been suggested to determine the function of the modulatory α-subunit Kv8.1. However, when we tested the NRD of Kv9.3, we found that the functional properties of chimeric Kv2.1 channels containing the NRD of Kv9.3 (Kv2.1NRD) did not resemble those of Kv2.1/Kv9.3 heteromers, thus questioning the role of the NRD in Kv9 subunits. A further region of interest is a PXP motif in the sixth transmembrane segment. This motif is conserved among all α-subunits of the Kv1, Kv2, Kv3, and Kv4 subfamilies, whereas the second proline is not conserved in any modulatory α-subunit. Exchanging this proline in Kv2.1 for the corresponding residue of Kv9.3 resulted in channels (Kv2.1-P410T) that show all hallmarks of the regulation of Kv2.1 by Kv9.3. The effect prevailed in heteromeric channels following co-expression of Kv2.1-P410T with Kv2.1. These data suggest that the alteration of the PXP motif is an important determinant of the regulatory function of modulatory α-subunits. Voltage-gated potassium (Kv) channels containing α-subunits of the Kv2 subfamily mediate delayed rectifier currents in excitable cells. Channels formed by Kv2.1 α-subunits inactivate from open- and closed states with both forms of inactivation serving different physiological functions. Here we show that open- and closed-state inactivation of Kv2.1 can be distinguished by the sensitivity to intracellular tetraethylammonium and extracellular potassium and lead to the same inactivated conformation. The functional properties of Kv2.1 are regulated by its association with modulatory α-subunits (Kv5, Kv6, Kv8, and Kv9). For instance, Kv9.3 changes the state preference of Kv2.1 inactivation by accelerating closed-state inactivation and inhibiting open-state inactivation. An N-terminal regulatory domain (NRD) has been suggested to determine the function of the modulatory α-subunit Kv8.1. However, when we tested the NRD of Kv9.3, we found that the functional properties of chimeric Kv2.1 channels containing the NRD of Kv9.3 (Kv2.1NRD) did not resemble those of Kv2.1/Kv9.3 heteromers, thus questioning the role of the NRD in Kv9 subunits. A further region of interest is a PXP motif in the sixth transmembrane segment. This motif is conserved among all α-subunits of the Kv1, Kv2, Kv3, and Kv4 subfamilies, whereas the second proline is not conserved in any modulatory α-subunit. Exchanging this proline in Kv2.1 for the corresponding residue of Kv9.3 resulted in channels (Kv2.1-P410T) that show all hallmarks of the regulation of Kv2.1 by Kv9.3. The effect prevailed in heteromeric channels following co-expression of Kv2.1-P410T with Kv2.1. These data suggest that the alteration of the PXP motif is an important determinant of the regulatory function of modulatory α-subunits. voltage-gated potassium N-terminal regulatory domain tetraethylammonium Voltage-gated potassium (Kv)1 channels form the most diverse class in the ion channel superfamily, giving rise to a large variety of currents, the kinetics of which are shaped to the requirements of their physiological function (1Hille B. Ionic Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Sunderland, MA2001Google Scholar). They are composed of four α-subunits, each containing six transmembrane segments (S1–S6), arranged around a central potassium-selective pore (2MacKinnon R. Nature. 1991; 350: 232-235Crossref PubMed Scopus (768) Google Scholar). The ability of different α-subunits to form heteromeric channels increases the diversity of K+ currents in native cells (3Christie M.J. North R.A. Osborne P.B. Douglass J. Adelman J.P. Neuron. 1990; 4: 405-411Abstract Full Text PDF PubMed Scopus (210) Google Scholar, 4Isacoff E.Y. Jan Y.N. Jan L.Y. Nature. 1990; 345: 530-534Crossref PubMed Scopus (379) Google Scholar, 5Ruppersberg J.P. Schroter K.H. Sakmann B. Stocker M. Sewing S. Pongs O. Nature. 1990; 345: 535-537Crossref PubMed Scopus (344) Google Scholar). Modulatory α-subunits constitute a group of proteins that are unable to build functional channels by themselves. They associate with Kv2 α-subunits forming heteromeric channels that activate, deactivate, inactivate, and recover from inactivation differently from homomeric Kv2 channels (6Kerschensteiner D. Stocker M. Biophys. J. 1999; 77: 248-257Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 7Patel A.J. Lazdunski M. Honore E. EMBO J. 1997; 16: 6615-6625Crossref PubMed Scopus (268) Google Scholar, 8Salinas M. de Weille J. Guillemare E. Lazdunski M. Hugnot J.P. J. Biol. Chem. 1997; 272: 8774-8780Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 9Salinas M. Duprat F. Heurteaux C. Hugnot J.P. Lazdunski M. J. Biol. Chem. 1997; 272: 24371-24379Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 10Kramer J.W. Post M.A. Brown A.M. Kirsch G.E. Am. J. Physiol. 1998; 274: C1501-C1510Crossref PubMed Google Scholar, 11Castellano A. Chiara M.D. Mellstrom B. Molina A. Monje F. Naranjo J.R. Lopez-Barneo J. J. Neurosci. 1997; 17: 4652-4661Crossref PubMed Google Scholar, 12Ottschytsch N. Raes A. Van Hoorick D. Snyders D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7986-7991Crossref PubMed Scopus (136) Google Scholar). The group of mammalian modulatory α-subunits so far consists of Kv5.1, Kv6.1–6.4, Kv8.1–2, and Kv9.1–9.3 (7Patel A.J. Lazdunski M. Honore E. EMBO J. 1997; 16: 6615-6625Crossref PubMed Scopus (268) Google Scholar, 10Kramer J.W. Post M.A. Brown A.M. Kirsch G.E. Am. J. Physiol. 1998; 274: C1501-C1510Crossref PubMed Google Scholar, 11Castellano A. Chiara M.D. Mellstrom B. Molina A. Monje F. Naranjo J.R. Lopez-Barneo J. J. Neurosci. 1997; 17: 4652-4661Crossref PubMed Google Scholar, 12Ottschytsch N. Raes A. Van Hoorick D. Snyders D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7986-7991Crossref PubMed Scopus (136) Google Scholar, 13Hugnot J.P. Salinas M. Lesage F. Guillemare E. de Weille J. Heurteaux C. Mattei M.G. Lazdunski M. EMBO J. 1996; 15: 3322-3331Crossref PubMed Scopus (108) Google Scholar, 14Stocker M. Kerschensteiner D. Biochem. Biophys. Res. Commun. 1998; 248: 927-934Crossref PubMed Scopus (30) Google Scholar, 15Stocker M. Hellwig M. Kerschensteiner D. J. Neurochem. 1999; 72: 1725-1734Crossref PubMed Scopus (46) Google Scholar). Their selective association with Kv2 α-subunits is guided by an intracellular N-terminal domain (15Stocker M. Hellwig M. Kerschensteiner D. J. Neurochem. 1999; 72: 1725-1734Crossref PubMed Scopus (46) Google Scholar, 16Post M.A. Kirsch G.E. Brown A.M. FEBS Lett. 1996; 399: 177-182Crossref PubMed Scopus (82) Google Scholar) initially identified in α-subunits of the subfamilies Kv1-Kv4 and named T1 for its role in tetramerization and subunit segregation (17Li M. Jan Y.N. Jan L.Y. Science. 1992; 257: 1225-1230Crossref PubMed Scopus (395) Google Scholar, 18Shen N.V. Chen X. Boyer M.M. Pfaffinger P.J. Neuron. 1993; 11: 67-76Abstract Full Text PDF PubMed Scopus (200) Google Scholar, 19Kreusch A. Pfaffinger P.J. Stevens C.F. Choe S. Nature. 1998; 392: 945-948Crossref PubMed Scopus (270) Google Scholar). We previously described the functional properties of heteromeric channels arising from the co-expression of Kv2.1 with Kv9.3 (Kv2.1/Kv9.3) (6Kerschensteiner D. Stocker M. Biophys. J. 1999; 77: 248-257Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Channel activation and deactivation were slowed, and their equilibrium shifted to hyperpolarized potentials when compared with homomeric Kv2.1 channels. Moreover, Kv9.3 changed the state dependence of Kv2.1 inactivation. Kv2 α-subunits have been identified as an important component of delayed rectifier currents in a variety of excitable cells where they participate in action potential repolarization, regulation of the firing frequency, and setting the resting membrane potential (20Du J. Haak L.L. Phillips-Tansey E. Russell J.T. McBain C.J. J. Physiol. 2000; 522: 19-31Crossref PubMed Scopus (165) Google Scholar, 21Blaine J.T. Ribera A.B. J. Neurosci. 2001; 21: 1473-1480Crossref PubMed Google Scholar, 22Quattrocki E.A. Marshall J. Kaczmarek L.K. Neuron. 1994; 12: 73-86Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 23Schultz J.H. Volk T. Ehmke H. Circ. Res. 2001; 88: 483-490Crossref PubMed Scopus (29) Google Scholar, 24Smirnov S.V. Beck R. Tammaro P. Ishii T. Aaronson P.I. J. Physiol. 2002; 538: 867-878Crossref PubMed Scopus (56) Google Scholar, 25Xu H. Barry D.M. Li H. Brunet S. Guo W. Nerbonne J.M. Circ. Res. 1999; 85: 623-633Crossref PubMed Scopus (143) Google Scholar). Inactivation of Kv2 channels reduces currents through these channels and thus regulates membrane excitability. Based on a model adapting the Monod-Wyman-Changeux model for allosteric proteins (26Monod J. Wyman J. Changeux J.-P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6184) Google Scholar) to ion channels, Kv2.1 has been suggested to inactivate from open and closed states (27Klemic K.G. Shieh C.C. Kirsch G.E. Jones S.W. Biophys. J. 1998; 74: 1779-1789Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The maximum of inactivation in this description was assigned to the last of five closed states passed by opening channels, which is linked with the open state through a transition with a voltage-independent on rate and a voltage-dependent off rate (27Klemic K.G. Shieh C.C. Kirsch G.E. Jones S.W. Biophys. J. 1998; 74: 1779-1789Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Here we refer to inactivation from this last closed and from open states as open-state inactivation. The term closed-state inactivation indicates inactivation from proximal states in the activation pathway that are separated from open states by voltage-dependent transitions. For Kv2.1 open-state inactivation is fast, and closed-state inactivation slow. On the contrary, heteromeric Kv2.1/Kv9.3 channels exhibit slow open- and fast closed-state inactivation. This shift to preferential closed-state inactivation has been referred to as a change in the state dependence of inactivation (6Kerschensteiner D. Stocker M. Biophys. J. 1999; 77: 248-257Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Preferential closed-state inactivation has been suggested to participate in the control of membrane excitability by modulating repetitive firing and back-propagation of action potentials in neurons as well as the repolarization of cardiac action potentials (28Johnston D. Hoffman D.A. Magee J.C. Poolos N.P. Watanabe S. Colbert C.M. Migliore M. J. Physiol. 2000; 525: 75-81Crossref PubMed Scopus (215) Google Scholar, 29Beck E.J. Bowlby M. An W.F. Rhodes K.J. Covarrubias M. J. Physiol. 2002; 538: 691-706Crossref PubMed Scopus (117) Google Scholar). Here we describe a pharmacological strategy to distinguish between open- and closed-state inactivation of Kv2.1 and show that both inactivation pathways lead to the same conformation. Moreover, we investigate the structural determinants underlying the regulation of Kv2.1 by the modulatory α-subunit Kv9.3. An N-terminal regulatory domain (NRD) originally characterized in Kv8.1 (30Chiara M.D. Monje F. Castellano A. Lopez-Barneo J. J. Neurosci. 1999; 19: 6865-6873Crossref PubMed Google Scholar) and suggested to govern its function was identified also in Kv9.3. However, the functional properties of chimeric Kv2.1 channels containing the NRD of Kv9.3 differed from those of Kv2.1/Kv9.3 heteromers, thereby questioning the importance of NRD in Kv9 subunits. On the contrary, we show that a single amino acid in the distal part of the pore-lining S6 determines the regulatory properties of Kv9.3. All of the DNA manipulations were carried out using standard recombinant DNA techniques (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The cDNAs coding for Kv2.1 (DRK1) and Kv9.3 used in this study were identical to the ones described previously (6Kerschensteiner D. Stocker M. Biophys. J. 1999; 77: 248-257Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 14Stocker M. Kerschensteiner D. Biochem. Biophys. Res. Commun. 1998; 248: 927-934Crossref PubMed Scopus (30) Google Scholar). Point mutations were introduced into Kv2.1 cDNA using QuikChange (Stratagene, La Jolla, CA). The chimera Kv2.1NRD was generated by replacing amino acids 127–181 of Kv2.1 by the NRD domain of Kv9.3 (amino acids 111–177). Amplification by low copy PCR (15 cycles) with the polymerase Pfu (Promega, Madison, WI) was used to generate one fragment of both Kv2.1 and Kv9.3. These fragments were then combined via a common introduced silent EcoRI recognition site and ligated into an appropriately cut Kv2.1. For all mutated or chimeric constructs, the sequence of the complete channel subunit was verified by sequencing with a BigDye terminator cycle sequencing kit and an ABI377 DNA sequencer (Applied Biosystems). Following linearization capped cRNAs were synthesized in vitro with mMessage mMachine (Ambion, Austin, TX). Isolation of oocytes (stages V and VI) from Xenopus laevis and cRNA injection were performed as described previously (32Stuhmer W. Methods Enzymol. 1992; 207: 319-339Crossref PubMed Scopus (262) Google Scholar). Whole cell currents were recorded 1–4 days after injection under two-electrode voltage-clamp control, using a Turbo TEC-10CD amplifier (NPI-Elektronik, Tamm, Germany). Intracellular electrodes had resistances of 0.3–0.8 MΩ when filled with 2 m KCl. The standard bath solution was normal frog Ringer containing 115 mm NaCl, 2.5 mm KCl, 1.8 mmCaCl2, 10 mm HEPES-NaOH (pH 7.2). In experiments in which KCl concentrations were raised, NaCl concentrations were lowered so that the sum of KCl an NaCl remained constant. Recordings testing the effect of internal TEA could not be performed in inside out patches because of fast rundown of Kv2.1 currents in this configuration (33Immke D. Wood M. Kiss L. Korn S.J. J. Gen. Physiol. 1999; 113: 819-836Crossref PubMed Scopus (74) Google Scholar). 2D. Kerschensteiner, F. Monje, and M. Stocker, unpublished observation. We therefore injected TEA into oocytes using a glass pipette filled with 105 mmKCl, 10 mm TEACl, 2.5 mm NaCl, 1.8 mm CaCl2, 10 mm HEPES-NaOH (pH 7.2). The currents were low pass filtered at 0.7–1 kHz (−3dB) and sampled at 3–5 kHz. All of the experiments were carried out at room temperature (20–22 °C). Data acquisition and analysis were performed with the Pulse+PulseFit software package (HEKA Elektronik, Lambrecht, Germany), EXCEL (Microsoft), and IGOR (Wavemetrics). Boltzman functions of the typePO/PO,max = Offset + 1/(1 + exp(V12 −Vm)/a) were used to fit steady-state activation. The data are given as the means ± S.E., with n specifying the number of independent experiments. Statistical significance was evaluated using a two-tailed Student's t test. The delayed rectifier potassium channel Kv2.1 (34Frech G.C. VanDongen A.M. Schuster G. Brown A.M. Joho R.H. Nature. 1989; 340: 642-645Crossref PubMed Scopus (358) Google Scholar) has previously been suggested to inactivate from open and closed states. This proposal was based on kinetic analyses (27Klemic K.G. Shieh C.C. Kirsch G.E. Jones S.W. Biophys. J. 1998; 74: 1779-1789Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and on the observation that co-expressions of different modulatory α-subunits with Kv2.1 have opposite effects on inactivation occurring at high and low open probabilities (6Kerschensteiner D. Stocker M. Biophys. J. 1999; 77: 248-257Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 10Kramer J.W. Post M.A. Brown A.M. Kirsch G.E. Am. J. Physiol. 1998; 274: C1501-C1510Crossref PubMed Google Scholar). Throughout this study open- and closed-state inactivation were measured using the pulse protocols PO inact andPC inact, respectively. ForPO inact, the membrane was depolarized for 32 s to potentials of high open probability. Time constants (τinact) derived from mono-exponential functions fit to the decay of the resulting outward currents were used to assess open-state inactivation (○ and ● in Figs.1, E and F,4D, and 5, E and F). ForPC inact, 300-ms test pulses to +40 mV were given at the beginning (P1) and end (Pn) of conditioning pulses of increasing length to potentials of low open probability. The ratio of currents elicited by the test pulses (IPn/IP1) is proportional to the number of channels that did not inactivate during the conditioning pulse and declines with increasing length of the conditioning pulse. Time constants (τinact) derived from mono-exponential functions fit to this decline were used to quantify closed-state inactivation (■ and ▪ in Figs. 1, E andF, 4D, and 5, E and F). For potentials of intermediate open probability both pulse protocols were used to measure inactivation occurring from both open and closed states (▵ and ▴ in Figs. 1, E and F, 4D, and 5, E and F).Figure 4The involvement of S6 in the regulatory function of Kv9.3.A, alignment of the last transmembrane segment (S6) for one representative member from each Kv subfamily. Kv subunits were assigned into subfamilies following the guidelines of the HUGO Gene Nomenclature Committee (www.gene.ucl.ac.uk/nomenclature/genefamily/KCN.shtml) B, currents through Kv2.1 and Kv2.1-P410T elicited by a 32-s depolarizing pulse to +40 mV (PO inact). To determine voltage dependence of activation (inset) for Kv2.1 (○) and Kv2.1-P410T (●), 300-ms pulses from −80 to +70 mV were applied in 10-mV increments. Subsequently, the voltage was clamped to −40 mV, and the initial current in this segment was estimated from a mono-exponential fit to its decay. The relative open probabilities (PO/PO, max) derived from the initial currents were plotted against the voltage of the conditioning pulses and fit with a Boltzmann function (n = 9–10). C, closed-state inactivation of Kv2.1 and Kv2.1-P410T measured by the pulse protocol illustrated (PC inact, Vinact = −40 mV). The currents in B and C were scaled to their maximum. D, time constants of inactivation of Kv2.1 (open symbols) and Kv2.1-P410T (filled symbols) measured by PO inact (○ and ●),PC inact (■ and ▪), or both (▵ and ▴), plotted as a function of the voltage of the inactivating pulse. The data in D are represented as the means ± S.E. (n = 4–10).View Large Image Figure ViewerDownload (PPT)Figure 5The pharmacological profile of Kv2.1-P410T corroborates preferential closed-state inactivation.A, currents through Kv2.1-P410T elicited by 32-s depolarizing pulse to +40 mV (PO inact) in the presence (+TEAi) or absence (−TEAi) of intracellular TEA. The currents were scaled to their maximum.B, currents evoked as in A with 2.5 or 115 mm K+ in the extracellular solution.C, closed-state inactivation of Kv2.1-P410T measured byPC inact (Vinact = −40 mV) in the presence (+TEAi) or absence (−TEAi) of intracellular TEA. D, recording as in C, with 2.5 or 115 mmK+ in the extracellular solution. E andF, time constants of inactivation evoked byPO inact (○ and ●),PC inact (■ and ▪), or both (▵ and ▴), plotted as a function of the voltage of the inactivating pulse.E, measurements in the presence (filled symbols) or absence (open symbols) of internal TEA. F, measurements with 2.5 mm (open symbols) or 115 mm (filled symbols) K+ in the extracellular solution. The data in E and F are presented as the means ± S.E. (n = 4–10).View Large Image Figure ViewerDownload (PPT) Presently our knowledge about the molecular determinants of these different inactivation pathways and the regulation by modulatory α-subunits is scarce. To have an additional tool for their analysis, we first investigated whether open- and closed-state inactivation can be separated pharmacologically. Kv2.1 channels were expressed inXenopus oocytes, and currents were measured under two-electrode voltage clamp conditions. The quaternary ammonium ion TEA, which blocks Kv2.1 channels (34Frech G.C. VanDongen A.M. Schuster G. Brown A.M. Joho R.H. Nature. 1989; 340: 642-645Crossref PubMed Scopus (358) Google Scholar, 35Ikeda S.R. Korn S.J. J. Physiol. 1995; 486: 267-272Crossref PubMed Scopus (43) Google Scholar), has been shown to slow C- (36Grissmer S. Cahalan M. Biophys. J. 1989; 55: 203-206Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 37Molina A. Castellano A.G. Lopez-Barneo J. J. Physiol. 1997; 499: 361-367Crossref PubMed Scopus (59) Google Scholar) and N-type inactivation (38Choi K.L. Aldrich R.W. Yellen G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5092-5095Crossref PubMed Scopus (395) Google Scholar, 39Zhou M. Morais-Cabral J.H. Mann S. MacKinnon R. Nature. 2001; 411: 657-661Crossref PubMed Scopus (494) Google Scholar) of other Kv channels when applied to the extra- or intracellular side of the channels, respectively. Therefore, we tested whether TEA was affecting Kv2.1 inactivation when applied from either side of the membrane. After the current response was stable for at least 5 min, TEA (10 mmsolution in high K+ Ringer) was injected through a third pipette impaled into the oocyte until approximately 50% of the initial current was blocked (55.3 ± 1.2%, n = 16). This intracellular application of TEA markedly slowed the rate of open-state inactivation (Fig. 1A, Vinact = +40 mV; E, ○ and ●; p < 0.02). On the other hand, closed-state inactivation was not altered significantly by internal TEA (Fig. 1C; Vinact = −30 mV; E, ■ and ▪; p > 0.6). Accordingly, Fig. 1E shows that the effect of intracellular TEA increases with more pronounced depolarizations, corresponding to increasing open probabilities of the channels. In agreement with previous publications, extracellular TEA did not affect open- or closed-state inactivation of Kv2.1 (data not shown and Ref. 27Klemic K.G. Shieh C.C. Kirsch G.E. Jones S.W. Biophys. J. 1998; 74: 1779-1789Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Additionally, we tested the influence of elevated extracellular [K+], a condition known to slow C-type inactivation of other Kv channels (40DeCoursey T.E. J. Gen. Physiol. 1990; 95: 617-646Crossref PubMed Scopus (44) Google Scholar, 41Levy D.I. Deutsch C. Biophys. J. 1996; 70: 798-805Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 42Baukrowitz T. Yellen G. Neuron. 1995; 15: 951-960Abstract Full Text PDF PubMed Scopus (326) Google Scholar). Elevation of the external [K+] from 2.5 to 115 mm significantly accelerated open-state inactivation. (Fig. 1B,Vinact = +40 mV; F, ○ and ●;p < 0.01). On the contrary, closed-state inactivation was decelerated when external [K+] was increased (Fig.1D, Vinact = −30 mV; F, ■ and ▪;p < 0.02). Taken together, these experiments demonstrate that the two inactivation pathways for Kv2.1 can be separated pharmacologically. Open-state inactivation is inhibited by intracellular TEA and accelerated by elevated external [K+], whereas closed-state inactivation is insensitive to intracellular TEA and inhibited by elevated external [K+]. The different sensitivities of open- and closed-state inactivation of Kv2.1 to internal TEA and external [K+] could arise by either of two mechanisms. First, inactivation from open and closed states could lead to different inactivated conformations of Kv2.1 from which recovery should differ, or second, they could represent different transitions that start from open or closed channels, respectively, but lead to the same inactivated conformation. In this case recovery should be independent from the way inactivation occurred. To discriminate between the two possibilities, recovery from open- and closed-state inactivation of Kv2.1 was analyzed. As illustrated in the Fig.2A, following a 300-ms pulse to +40 mV (P1) channels were inactivated by clamping the voltage for 40 s at either +40 mV (open-state inactivation) or −40 mV (closed-state inactivation) and a second 300-ms pulse to +40 mV (P2) was given to measure the degree of inactivation. Channels were then recovered from inactivation at hyperpolarized potentials (−80 to −120 mV) for increasing time intervals before applying another 300-ms pulse to +40 mV (Pn). The number of channels that recover during this period is proportional to the ratio of currents elicited by the short pulses (IPn −IP2/IP1 −IP2) and increases with increasing time spent at the recovery potential. Time constants derived from mono-exponential functions fit to this process were used to quantify recovery. They were indistinguishable between recovery from open- (Rec −Io) and closed-state inactivation (Rec −Ic) at all potentials tested (Fig.2B, p > 0.3). Moreover, elevating the extracellular [K+] accelerated recovery to a rate that was identical for open- and closed-state inactivation (Fig.2C, p > 0.2). These observations support the idea that open- and closed-state inactivation of Kv2.1 are separate transitions leading to the same inactivated conformation. We previously characterized the modulatory α-subunit Kv9.3, which forms heteromeric channels with Kv2.1 and changes the preferred state from which inactivation occurs (6Kerschensteiner D. Stocker M. Biophys. J. 1999; 77: 248-257Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Although Kv2.1 inactivates fast from open and slowly from closed states, heteromeric Kv2.1/Kv9.3 channels show little open- but fast and complete closed-state inactivation (Fig. 3, A and B) (6Kerschensteiner D. Stocker M. Biophys. J. 1999; 77: 248-257Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The molecular determinants of this regulation are unknown. The inhibition of open-state inactivation of Kv2.1 by the modulatory α-subunit Kv8.1 has been attributed to a segment of 59 amino acids preceding the first transmembrane segment (30Chiara M.D. Monje F. Castellano A. Lopez-Barneo J. J. Neurosci. 1999; 19: 6865-6873Crossref PubMed Google Scholar). A comparison of this region, termed NRD (30Chiara M.D. Monje F. Castellano A. Lopez-Barneo J. J. Neurosci. 1999; 19: 6865-6873Crossref PubMed Google Scholar), with the respective region in Kv9.3 showed little sequence identity. To test whether the proposed function of this domain was conserved, chimeric Kv2.1 channels containing the NRD of Kv9.3 (Kv2.1NRD) were constructed (Fig. 3C). Steady-state activation of Kv2.1NRD was shifted in the depolarized direction with respect to Kv2.1 (Kv2.1,V12 = 3 ± 1 mV (n = 9); Kv2.1NRD, V12 = 12 ± 2 mV, (n = 12) (Fig. 3D) in contrast to what has been observed for Kv2.1/Kv9.3 heteromers, which opened at more hyperpolarized potentials than Kv2.1 (6Kerschensteiner D. Stocker M. Biophys. J. 1999; 77: 248-257Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). For Kv2.1NRD, as for Kv2.1/Kv9.3 heteromers, the kinetics of activation and deactivation were slowed (data not shown), and open-state inactivation was reduced. Accordingly, at the end of 32-s depolarizing pulses to +40 mV only 34 ± 0.4% (n = 6) of the Kv2.1NRDcurrent was inactivated, compared with 84.9 ± 1.8% (n = 24) of the Kv2.1 current (Fig. 3, E andG). However, Kv2.1NRD channels did not show the prominent closed-state inactivation that is the hallmark of Kv2.1/Kv9.3 heteromers (Fig. 3B) (6Kerschensteiner D. Stocker M. Biophys. J. 1999; 77: 248-257Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). On the contrary, they display less closed-state inactivation than Kv2.1. Thus, following 32 s at −30 mV, 84 ± 1.1% (n = 6) of the initial Kv2.1NRD current was left compared with 59.7 ± 5.5%, (n = 16) for Kv2.1 (Fig. 3, F andG). In conclusion, the NRD of Kv9.3, rather than shifting the maximum of inactivation from open to closed states, inhibits both inactivation pathways. Hence, another region of Kv9.3 must participate in the regulation of Kv2.1. To find such alternative regions we compared the amino acid sequences of all of modulatory α-subunits (Kv5.1, Kv6.1–6.4, Kv8.1–8.2, and Kv9.1–9.3) with the sequences of Kv1, Kv2, Kv3, and Kv4 α-subunits and thus identified the sixth transmembrane segment (S6) as a region of interest. Fig.4A shows an alignment of the S6 in which one representative member of each subfamily was included. The second proline of a PXP motif (Fig. 4A,arrow) is conserved among all 17 α-subunits of the Kv1–Kv4 subfamilies and is replaced by serine, threonine, alanine, or histidine in all 10 modulatory α-subunits. To test the hypothesis that the alteration of this PXP motif participates in the regulatory function of Kv9.3, we mutated the proline at position 410 in Kv2.1 to the corresponding threonine of Kv9.3. The resulting α-subunit (Kv2.1-P410T) formed functional channels inXenopus oocytes. Steady-state activation of Kv2.1-P410T was shifted 12 mV toward hyperpolarized potentials compared with Kv2.1 (Kv2.1, V12 = 3 ± 1 mV, n = 9; Kv2.1-P410T, V12 = −9 ± 2 mV, n = 10; Fig. 4B, inset), and activation and deactivation kinetics decelerated, similar to what has been observed for heteromeric Kv2.1/Kv9.3 channels (6Kerschensteiner D. Stocker M. Bio
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