Transfer of rapid inactivation and sensitivity to the class III antiarrhythmic drug E-4031 from HERG to M-eag channels

Autor: Gail A. Robertson, Ian M. Herzberg, Matthew C. Trudeau
Rok vydání: 1998
Předmět:
Models
Molecular
congenital
hereditary
and neonatal diseases and abnormalities
ERG1 Potassium Channel
Potassium Channels
Protein Conformation
Pyridines
Physiology
Recombinant Fusion Proteins
Molecular Sequence Data
hERG
Gating
Membrane Potentials
Mice
chemistry.chemical_compound
Piperidines
Transcriptional Regulator ERG
Potassium Channel Blockers
Animals
Humans
Point Mutation
cardiovascular diseases
Amino Acid Sequence
Cation Transport Proteins
Tetraethylammonium
Sequence Homology
Amino Acid
biology
Chemistry
Cardiac action potential
Original Articles
Ether-A-Go-Go Potassium Channels
Potassium channel
DNA-Binding Proteins
Transplantation
Transmembrane domain
Biochemistry
Potassium Channels
Voltage-Gated
Mutagenesis
Site-Directed
Trans-Activators
Biophysics
biology.protein
E-4031
Anti-Arrhythmia Agents
Ion Channel Gating
Sequence Alignment
Zdroj: The Journal of Physiology. 511:3-14
ISSN: 0022-3751
DOI: 10.1111/j.1469-7793.1998.003bi.x
Popis: The gating behaviour and pharmacological sensitivity of HERG are remarkably different from the corresponding properties of M-eag, a structurally similar member of the Eag family of potassium channels. In contrast to HERG, M-eag exhibits no apparent inactivation and little rectification, and is insensitive to the class III antiarrhythmic drug E-4031. We generated chimeric channels of HERG and M-eag sequences and made point mutations to identify the region necessary for rapid inactivation in HERG. This region includes the P region and half of the S6 putative transmembrane domain, including sites not previously associated with inactivation and rectification in HERG. Transfer of a small segment of the HERG polypeptide to M-eag, consisting largely of the P region and part of the S6 transmembrane domain, is sufficient to confer rapid inactivation and E-4031 sensitivity to M-eag. This region differs from the corresponding region in M-eag by only fifteen residues. Previous hypotheses that rapid inactivation of HERG channels occurs by a C-type inactivation mechanism are supported by the parallel effects on rates of HERG inactivation and Shaker C-type inactivation by a series of mutations at two equivalent sites in the polypeptide sequences. In addition to sites homologous to those previously described for C-type inactivation in Shaker, inactivation in HERG involves a residue in the upstream P region not previously associated with C-type inactivation. Although this site is equivalent to one implicated in P-type inactivation in Kv2.1 channels, our data are most consistent with a single, C-type inactivation mechanism. The human ether-a-go-go-related gene (HERG) and the murine ether-a-go-go gene (M-eag) were isolated based on their homology to the Drosophila ether-a-go-go gene (eag; Warmke & Ganetzky, 1994). HERG and M-eag are members of distinct subfamilies within the Eag family which, although only distantly related to the Shaker family of potassium channel genes, encode polypeptides that share important structural features (Warmke et al. 1991). For example, members of the Eag family of potassium channels contain six putative membrane spanning domains, an S4 region shown to mediate voltage-dependent gating in Shaker channels (Papazian et al. 1991; Liman et al. 1991; Logothetis et al. 1992; Schoppa et al. 1992; Bezanilla et al. 1994; Perozo et al. 1994; Zagotta et al. 1994) and a P region highly conserved with those sequences governing ion permeation in Shaker and related channels (MacKinnon & Miller, 1989; MacKinnon et al. 1990; MacKinnon & Yellen, 1990; Yellen et al. 1991; Hartmann et al. 1991; Kavanaugh et al. 1991). Among the superfamily of S4-containing potassium channels, HERG is distinguished by its linkage to a form of congenital long QT syndrome (Curran et al. 1995) and its identification with the native IKr (Sanguinetti et al. 1995; Trudeau et al. 1995), a current important in the repolarization of the cardiac action potential (Sanguinetti & Jurkiewicz, 1990). Expression studies in Xenopus oocytes indicate that HERG and M-eag channels are both potassium selective and gated by voltage in approximately the same range (Trudeau et al. 1995; Robertson et al. 1996), consistent with a high degree of similarity in their primary structure (49 % in the hydrophobic core). Despite these similarities, M-eag exhibits neither the fast inactivation nor pronounced inward rectification characteristic of HERG and cardiac IKr. The term ‘inward rectification’ describes the reduction in outward HERG current amplitudes at positive voltages resulting in a region of negative slope conductance in the I-V relation. Inward rectification is due to a fast inactivation mechanism that limits the occupancy of the open state at positive potentials (Shibasaki, 1987; Sanguinetti et al. 1995; Trudeau et al. 1995; Spector et al. 1996a). During an action potential, therefore, maximal current is achieved only after the membrane begins to repolarize, as channels recover from the inactivated state and pass through the open state prior to closing (Zhou et al. 1998). The inactivation underlying inward rectification in HERG is similar in many ways to C-type inactivation originally described in Shaker channels (Hoshi et al. 1991). For example, external tetraethylammonium (TEA; Smith et al. 1996) and elevated external potassium (Wang et al. 1996) reduce the rate of inactivation in HERG channels. In addition, inactivation is slowed by a serine-to-alanine mutation in the pore region (S631A; Schonherr & Heinemann, 1996; Suessbrich et al. 1997) and eliminated by a double mutation involving this and one other site in the P region (G628C/S631C; Smith et al. 1996). These results are consistent with the slowing of C-type inactivation in Shaker channels by external TEA (Choi et al. 1991) and potassium (Lopez-Barneo et al. 1993), and by mutations at the equivalent position in the Shaker P region (T449; Lopez-Barneo et al. 1993; Schlief et al. 1996). In contrast to Shaker C-type inactivation, however, inactivation in HERG is inherently voltage dependent (Spector et al. 1996a; Wang et al. 1996). N-type inactivation is not essential for inward rectification, which is maintained when the N terminus is deleted (Schonherr & Heinemann, 1996; Spector et al. 1996a). We examined the molecular basis of rapid inactivation in HERG using an approach that was independent of pre-existing models. We exploited the structural similarities between HERG and M-eag by creating a library of chimeric channels with an in vivo recombination method in E. coli. We mapped the position of each crossover and recorded currents from the chimeric channels expressed in Xenopus oocytes. By assaying for inward rectification, we mapped the region important for inactivation in HERG channels, identifying critical sites in the upstream P region, the downstream P region and the sixth transmembrane domain (S6). Transplantation of the entire P region and a portion of the S6 domain to M-eag is sufficient to confer inward rectification and inactivation, as well as sensitivity to the class III antiarrhythmic drug E-4031. Moreover, mutagenesis of residues within this region supports previous claims that C-type inactivation mediates inward rectification in HERG.
Databáze: OpenAIRE
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