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MinK subdomains that mediate modulation of and association with KvLQT1.

Tapper AR, George AL - J. Gen. Physiol. (2000)

Bottom Line: Currents derived from coexpression of KvLQT1 with MinK DeltaCterm were cadmium sensitive, suggesting that MinK DeltaCterm does associate with KvLQT1, but does not modulate gating.The results from this analysis indicate that MiRP1 cannot modulate KvLQT1 due to differences within the transmembrane domain.Our results allow us to identify the MinK subdomains that mediate KvLQT1 association and modulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

ABSTRACT
KvLQT1 is a voltage-gated potassium channel expressed in cardiac cells that is critical for myocardial repolarization. When expressed alone in heterologous expression systems, KvLQT1 channels exhibit a rapidly activating potassium current that slowly deactivates. MinK, a 129 amino acid protein containing one transmembrane-spanning domain modulates KvLQT1, greatly slowing activation, increasing current amplitude, and removing inactivation. Using deletion and chimeric analysis, we have examined the structural determinants of MinK effects on gating modulation and subunit association. Coexpression of KvLQT1 with a MinK COOH-terminus deletion mutant (MinK DeltaCterm) in Xenopus oocytes resulted in a rapidly activated potassium current closely resembling currents recorded from oocytes expressing KvLQT1 alone, indicating that this region is necessary for modulation. To determine whether MinK DeltaCterm was associated with KvLQT1, a functional tag (G55C) that confers susceptibility to partial block by external cadmium was engineered into the transmembrane domain of MinK DeltaCterm. Currents derived from coexpression of KvLQT1 with MinK DeltaCterm were cadmium sensitive, suggesting that MinK DeltaCterm does associate with KvLQT1, but does not modulate gating. To determine which MinK regions are sufficient for KvLQT1 association and modulation, chimeras were generated between MinK and the Na(+) channel beta1 subunit. Chimeras between MinK and beta1 could only modulate KvLQT1 if they contained both the MinK transmembrane domain and COOH terminus, suggesting that the MinK COOH terminus alone is not sufficient for KvLQT1 modulation, and requires an additional, possibly associative interaction between the MinK transmembrane domain and KvLQT1. To identify the MinK subdomains necessary for gating modulation, deletion mutants were designed and coexpressed with KvLQT1. A MinK construct with amino acid residues 94-129 deleted retained the ability to modulate KvLQT1 gating, identifying the COOH-terminal region critical for gating modulation. Finally, MinK/MiRP1 (MinK related protein-1) chimeras were generated to investigate the difference between these two closely related subunits in their ability to modulate KvLQT1. The results from this analysis indicate that MiRP1 cannot modulate KvLQT1 due to differences within the transmembrane domain. Our results allow us to identify the MinK subdomains that mediate KvLQT1 association and modulation.

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MinK Modulation of KvLQT1. (A) Two-electrode voltage-clamp recordings from Xenopus oocytes expressing either KvLQT1 (left) or KvLQT1+MinK (right). From a holding potential of −80 mV, oocytes were depolarized for 2 s to test potentials between +60 and −50 mV in 10-mV steps, followed by repolarization to −70 mV for 1 s. (B) Tail currents from oocytes expressing either KvLQT1 (left) or KvLQT1+MinK (right). Tail currents were elicited by a 4-s +40-mV prepulse, followed by repolarization to potentials between −40 and −100 mV in 10-mV steps. (C) Current–voltage relationship for KvLQT1 or KvLQT1+MinK. Currents were recorded after 2-s pulses at the given test potential. Error bars represent SEM (n = 5). (D) Normalized isochronal (t = 2 s) activation curve for five oocytes expressing either KvLQT1 or KvLQT1+MinK. The activation curves were derived from currents elicited by the activation protocol described in A. Experimental data points were fit with the equation 1/[1 + exp(V − V1/2)/k], which gave the following apparent V1/2 and slope factors: for KvLQT1: V1/2 = −27.4 ± 1.1 mV, k = 14.7 ± 0.66; and for KvLQT1+MinK: V1/2app = 29.4 ± 1.7 mV, k = 16.4 ± 1.6. Error bars represent SEM.
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Figure 1: MinK Modulation of KvLQT1. (A) Two-electrode voltage-clamp recordings from Xenopus oocytes expressing either KvLQT1 (left) or KvLQT1+MinK (right). From a holding potential of −80 mV, oocytes were depolarized for 2 s to test potentials between +60 and −50 mV in 10-mV steps, followed by repolarization to −70 mV for 1 s. (B) Tail currents from oocytes expressing either KvLQT1 (left) or KvLQT1+MinK (right). Tail currents were elicited by a 4-s +40-mV prepulse, followed by repolarization to potentials between −40 and −100 mV in 10-mV steps. (C) Current–voltage relationship for KvLQT1 or KvLQT1+MinK. Currents were recorded after 2-s pulses at the given test potential. Error bars represent SEM (n = 5). (D) Normalized isochronal (t = 2 s) activation curve for five oocytes expressing either KvLQT1 or KvLQT1+MinK. The activation curves were derived from currents elicited by the activation protocol described in A. Experimental data points were fit with the equation 1/[1 + exp(V − V1/2)/k], which gave the following apparent V1/2 and slope factors: for KvLQT1: V1/2 = −27.4 ± 1.1 mV, k = 14.7 ± 0.66; and for KvLQT1+MinK: V1/2app = 29.4 ± 1.7 mV, k = 16.4 ± 1.6. Error bars represent SEM.

Mentions: MinK modulates KvLQT1 gating by slowing activation and removing inactivation. In addition, MinK changes the rectification of KvLQT1 currents, although the mechanism of this is unclear. MinK also modulates KvLQT1 current amplitude by an unknown mechanism that may or may not be dependent on gating. Fig. 1 illustrates the principle effects of MinK on coexpressed KvLQT1 in Xenopus oocytes. Upon depolarization, oocytes expressing KvLQT1 alone exhibit a rapidly activating, slowly deactivating outward potassium current with characteristics similar to previously published data (Fig. 1 A, left; Barhanin et al. 1996; Sanguinetti et al. 1996). KvLQT1 tail currents display a “hook,” indicating that KvLQT1 inactivates to some extent (Fig. 1 B, left; Pusch et al. 1998; Tristani-Firouzi and Sanguinetti 1998). KvLQT1-induced currents exhibit a linear current–voltage relationship from −40 to +60 mV (Fig. 1 C). MinK dramatically modulates KvLQT1 gating, slowing activation (Fig. 1 A, right), removing inactivation (B, right), and shifting the voltage dependence of activation to more positive potentials (D). MinK-modulated currents have three- to fivefold greater current amplitudes than KvLQT1 alone (Fig. 1 C). Understanding how MinK associates with KvLQT1 and identifying subregions involved in gating modulation may provide insight into the mechanism by which MinK modulates KvLQT1.


MinK subdomains that mediate modulation of and association with KvLQT1.

Tapper AR, George AL - J. Gen. Physiol. (2000)

MinK Modulation of KvLQT1. (A) Two-electrode voltage-clamp recordings from Xenopus oocytes expressing either KvLQT1 (left) or KvLQT1+MinK (right). From a holding potential of −80 mV, oocytes were depolarized for 2 s to test potentials between +60 and −50 mV in 10-mV steps, followed by repolarization to −70 mV for 1 s. (B) Tail currents from oocytes expressing either KvLQT1 (left) or KvLQT1+MinK (right). Tail currents were elicited by a 4-s +40-mV prepulse, followed by repolarization to potentials between −40 and −100 mV in 10-mV steps. (C) Current–voltage relationship for KvLQT1 or KvLQT1+MinK. Currents were recorded after 2-s pulses at the given test potential. Error bars represent SEM (n = 5). (D) Normalized isochronal (t = 2 s) activation curve for five oocytes expressing either KvLQT1 or KvLQT1+MinK. The activation curves were derived from currents elicited by the activation protocol described in A. Experimental data points were fit with the equation 1/[1 + exp(V − V1/2)/k], which gave the following apparent V1/2 and slope factors: for KvLQT1: V1/2 = −27.4 ± 1.1 mV, k = 14.7 ± 0.66; and for KvLQT1+MinK: V1/2app = 29.4 ± 1.7 mV, k = 16.4 ± 1.6. Error bars represent SEM.
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Figure 1: MinK Modulation of KvLQT1. (A) Two-electrode voltage-clamp recordings from Xenopus oocytes expressing either KvLQT1 (left) or KvLQT1+MinK (right). From a holding potential of −80 mV, oocytes were depolarized for 2 s to test potentials between +60 and −50 mV in 10-mV steps, followed by repolarization to −70 mV for 1 s. (B) Tail currents from oocytes expressing either KvLQT1 (left) or KvLQT1+MinK (right). Tail currents were elicited by a 4-s +40-mV prepulse, followed by repolarization to potentials between −40 and −100 mV in 10-mV steps. (C) Current–voltage relationship for KvLQT1 or KvLQT1+MinK. Currents were recorded after 2-s pulses at the given test potential. Error bars represent SEM (n = 5). (D) Normalized isochronal (t = 2 s) activation curve for five oocytes expressing either KvLQT1 or KvLQT1+MinK. The activation curves were derived from currents elicited by the activation protocol described in A. Experimental data points were fit with the equation 1/[1 + exp(V − V1/2)/k], which gave the following apparent V1/2 and slope factors: for KvLQT1: V1/2 = −27.4 ± 1.1 mV, k = 14.7 ± 0.66; and for KvLQT1+MinK: V1/2app = 29.4 ± 1.7 mV, k = 16.4 ± 1.6. Error bars represent SEM.
Mentions: MinK modulates KvLQT1 gating by slowing activation and removing inactivation. In addition, MinK changes the rectification of KvLQT1 currents, although the mechanism of this is unclear. MinK also modulates KvLQT1 current amplitude by an unknown mechanism that may or may not be dependent on gating. Fig. 1 illustrates the principle effects of MinK on coexpressed KvLQT1 in Xenopus oocytes. Upon depolarization, oocytes expressing KvLQT1 alone exhibit a rapidly activating, slowly deactivating outward potassium current with characteristics similar to previously published data (Fig. 1 A, left; Barhanin et al. 1996; Sanguinetti et al. 1996). KvLQT1 tail currents display a “hook,” indicating that KvLQT1 inactivates to some extent (Fig. 1 B, left; Pusch et al. 1998; Tristani-Firouzi and Sanguinetti 1998). KvLQT1-induced currents exhibit a linear current–voltage relationship from −40 to +60 mV (Fig. 1 C). MinK dramatically modulates KvLQT1 gating, slowing activation (Fig. 1 A, right), removing inactivation (B, right), and shifting the voltage dependence of activation to more positive potentials (D). MinK-modulated currents have three- to fivefold greater current amplitudes than KvLQT1 alone (Fig. 1 C). Understanding how MinK associates with KvLQT1 and identifying subregions involved in gating modulation may provide insight into the mechanism by which MinK modulates KvLQT1.

Bottom Line: Currents derived from coexpression of KvLQT1 with MinK DeltaCterm were cadmium sensitive, suggesting that MinK DeltaCterm does associate with KvLQT1, but does not modulate gating.The results from this analysis indicate that MiRP1 cannot modulate KvLQT1 due to differences within the transmembrane domain.Our results allow us to identify the MinK subdomains that mediate KvLQT1 association and modulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

ABSTRACT
KvLQT1 is a voltage-gated potassium channel expressed in cardiac cells that is critical for myocardial repolarization. When expressed alone in heterologous expression systems, KvLQT1 channels exhibit a rapidly activating potassium current that slowly deactivates. MinK, a 129 amino acid protein containing one transmembrane-spanning domain modulates KvLQT1, greatly slowing activation, increasing current amplitude, and removing inactivation. Using deletion and chimeric analysis, we have examined the structural determinants of MinK effects on gating modulation and subunit association. Coexpression of KvLQT1 with a MinK COOH-terminus deletion mutant (MinK DeltaCterm) in Xenopus oocytes resulted in a rapidly activated potassium current closely resembling currents recorded from oocytes expressing KvLQT1 alone, indicating that this region is necessary for modulation. To determine whether MinK DeltaCterm was associated with KvLQT1, a functional tag (G55C) that confers susceptibility to partial block by external cadmium was engineered into the transmembrane domain of MinK DeltaCterm. Currents derived from coexpression of KvLQT1 with MinK DeltaCterm were cadmium sensitive, suggesting that MinK DeltaCterm does associate with KvLQT1, but does not modulate gating. To determine which MinK regions are sufficient for KvLQT1 association and modulation, chimeras were generated between MinK and the Na(+) channel beta1 subunit. Chimeras between MinK and beta1 could only modulate KvLQT1 if they contained both the MinK transmembrane domain and COOH terminus, suggesting that the MinK COOH terminus alone is not sufficient for KvLQT1 modulation, and requires an additional, possibly associative interaction between the MinK transmembrane domain and KvLQT1. To identify the MinK subdomains necessary for gating modulation, deletion mutants were designed and coexpressed with KvLQT1. A MinK construct with amino acid residues 94-129 deleted retained the ability to modulate KvLQT1 gating, identifying the COOH-terminal region critical for gating modulation. Finally, MinK/MiRP1 (MinK related protein-1) chimeras were generated to investigate the difference between these two closely related subunits in their ability to modulate KvLQT1. The results from this analysis indicate that MiRP1 cannot modulate KvLQT1 due to differences within the transmembrane domain. Our results allow us to identify the MinK subdomains that mediate KvLQT1 association and modulation.

Show MeSH
Related in: MedlinePlus