Shamgar L et al. (2008), KCNE1 constrains the voltage sensor of Kv7.1 K+...

XB-ART-37931

PLoS One 2008 Apr 09;34:e1943. doi: 10.1371/journal.pone.0001943.

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KCNE1 constrains the voltage sensor of Kv7.1 K+ channels.

Shamgar L , Haitin Y , Yisharel I , Malka E , Schottelndreier H , Peretz A , Paas Y , Attali B .

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Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K(+) channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs) and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K(+) channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions.

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Species referenced: Xenopus laevis
Genes referenced: arfgap1 kcna7 kcne1 kcne3 kcnq1

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	Figure 1. Summary of the tryptophan scan of Kv7.1 S4.(A) Sequence alignment of the S4 segment of human Kv7.1 with various Kv channels. (B) Impact of the perturbations projected onto a helical wheel diagram. The cut-off for significance was â¥ÎÎG0câ¥â¥1.5 kcal.molâ1. The red, blue and green circled mutated residues shift the gating equilibrium in favor of the closed, open and inactivated state, respectively. (C) Impact of the perturbations expressed as a bar graph along the S4 sequence. The color coding is as in B. The black bars correspond to residues whose perturbation is not significant (â¥ÎÎG0câ¥<1.5 kcal.molâ1).
	Figure 2. Mutations stabilizing Kv7.1 to the open state.(A) and (B) Representative current traces of WT and A226W, respectively. From a holding potential of â90 mV, the membrane was stepped for 3 s from â70 mV to +60 mV in 10 mV increments and then repolarized for 1.5 s to â60 mV to generate the tail currents. (C) and (D) Normalized conductance was plotted as a function of step voltages, for the mutants (black squares) A226W (nâ=â6) and V241W (nâ=â11), respectively, and compared to WT (nâ=â20) (open squares). The activation curves were fitted using one Boltzmann function. (E) Representative current traces of R231W. Membrane was stepped for 3 s from â140 mV to +60 mV in 20 mV increments and then repolarized for 1.5 s to â60 mV. (F) Current-voltage relations of R231W (nâ=â8) (black squares) and WT (open squares). Current density (pA/pF) was plotted as a function of step voltages.
	Figure 3. Mutations stabilizing Kv7.1 to the closed state.(A) and (B) Representative current traces of WT and L239W, respectively, recorded as in Figure 2 A. (C) and (D) Normalized conductance of the mutants (black squares) L239W (nâ=â5) and L236W (nâ=â10), respectively, compared to WT (open squares). (E) and (F) Representative current traces of R228W and I235W, respectively, recorded as in Figure 2 A. (G) Normalized conductance of R228W (nâ=â8) (black squares), compared to WT (open squares). (H) The time t1/2 needed to reach half-maximal current amplitude was determined for R228W, I235W and WT (nâ=â8â20).
	Figure 4. Mutations stabilizing KCNQ1 towards the inactivated state.(A) and (B) Representative current traces of WT and L233W and Q244W, respectively, recorded as in Figure 2 A. (C) Normalized conductance of L233W (nâ=â13) (black squares), compared to WT (open squares). (D) Percent of macroscopic inactivation of WT, Q244W and L233W (nâ=â7â20) as measured by the ratio between the sustained and the peak current amplitudes.
	Figure 5. Summary of the tryptophan scan of Kv7.1 S4 in the presence of KCNE1.The cut-off for significance was â¥ÎÎG0câ¥â¥1.5 kcal.molâ1. The red and blue bars of the mutated residues shift the gating equilibrium in favor of the closed and open state, respectively. The black bars correspond to residues whose perturbation is not significant (â¥ÎÎG0câ¥<1.5 kcal.molâ1).
	Figure 6. Effect of KCNE1 co-expression with mutant G229W.Representative current traces of WT IKS (A) and mutant G229W expressed without (B) or with KCNE1 (C). From a holding potential of â90 mV, the membrane was stepped for 3 s from â70 mV to +60 mV in 10 mV increments and then repolarized for 1.5 s to â60 mV to generate the tail currents. Conductance-voltage relations (D) and current-voltage relations (E) of WT Kv7.1 and mutant G229W co-expressed with KCNE1.
	Figure 7. Effect of KCNE1 co-expression with mutant R231W and I235W.Representative current traces of mutant R231W expressed without (A) or with KCNE1 (B). Conductance-voltage relations (C) and current-voltage relations (D) of WT Kv7.1 and mutant R231W co-expressed with KCNE1. Representative current traces of mutant I235W expressed without (E) or with KCNE1 (F). Conductance-voltage relations (G) and current-voltage relations (H) of WT Kv7.1 and mutant I235W co-expressed with KCNE1.
	Figure 8. Effect of KCNE1 co-expression with mutant R237W and R243W.Representative current traces of mutant R237W expressed without (A) or with KCNE1 (B). Conductance-voltage relations (C) and current-voltage relations (D) of WT Kv7.1 and mutant R237W co-expressed with KCNE1. Representative current traces of mutant R243W expressed without (E) or with KCNE1 (F). Conductance-voltage relations (G) and current-voltage relations (H) of WT Kv7.1 and mutant R243W co-expressed with KCNE1.
	Figure 9. Impact of KCNE1 expression on WT Kv7.1 and mutant R228C.(A) Representative trace of WT Kv7.1 coexpressed with WT KCNE1. (B) Effects of external Cu-Phen on mutant R228C. Oocytes were bathed in ND96 in the absence and presence of 100 ÂµM Cu-Phen. Shown are representative traces and current-voltage relations were determined as indicated. (C) Shown are representative traces and current-voltage relations of R228C+WT KCNE1 channels, when oocytes were bathed with ND96 in the absence of presence of 100 ÂµM Cu-Phen. Also shown, is the reversal by DTT of the current decrease produced by Cu-Phen. (D) Representative traces of R228C+WT KCNE1 channels, when oocytes were bathed with ND96 containing 100 ÂµM Cu-Phen. Currents were evoked by a train of step depolarization to +30 mV. Similar results have been obtained in 5 other cells.
	Figure 10. Models corresponding to the three-dimensional structure of Kv1.7 K+ channel and its auxiliary subunit KCNE1.(A) and (B), the closed state in top and side views, respectively. (C) and (D), the open state in top and side views, respectively. Ribbon diagrams of four differently colored identical subunits with the membrane embedded helical segment of KCNE1 in grey.

References [+] :

Abbott, A superfamily of small potassium channel subunits: form and function of the MinK-related peptides (MiRPs). 1998, Pubmed