Gage SD and Kobertz WR (2004), KCNE3 truncation mutants reveal a bipartite mod...

XB-ART-2662

J Gen Physiol 2004 Dec 01;1246:759-71. doi: 10.1085/jgp.200409114.

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KCNE3 truncation mutants reveal a bipartite modulation of KCNQ1 K+ channels.

Gage SD , Kobertz WR .

Abstract
The five KCNE genes encode a family of type I transmembrane peptides that assemble with KCNQ1 and other voltage-gated K(+) channels, resulting in potassium conducting complexes with varied channel-gating properties. It has been recently proposed that a triplet of amino acids within the transmembrane domain of KCNE1 and KCNE3 confers modulation specificity to the peptide, since swapping of these three residues essentially converts the recipient KCNE into the donor (Melman, Y.F., A. Domenech, S. de la Luna, and T.V. McDonald. 2001. J. Biol. Chem. 276:6439-6444). However, these results are in stark contrast with earlier KCNE1 deletion studies, which demonstrated that a COOH-terminal region, highly conserved between KCNE1 and KCNE3, was responsible for KCNE1 modulation of KCNQ1 (Tapper, A.R., and A.L. George. 2000 J. Gen. Physiol. 116:379-389.). To ascertain whether KCNE3 peptides behave similarly to KCNE1, we examined a panel of NH(2)- and COOH-terminal KCNE3 truncation mutants to directly determine the regions required for assembly with and modulation of KCNQ1 channels. Truncations lacking the majority of their NH(2) terminus, COOH terminus, or mutants harboring both truncations gave rise to KCNQ1 channel complexes with basal activation, a hallmark of KCNE3 modulation. These results demonstrate that the KCNE3 transmembrane domain is sufficient for assembly with and modulation of KCNQ1 channels and suggests a bipartite model for KCNQ1 modulation by KCNE1 and KCNE3 subunits. In this model, the KCNE3 transmembrane domain is active in modulation and overrides the COOH terminus'' contribution, whereas the KCNE1 transmembrane domain is passive and reveals COOH-terminal modulation of KCNQ1 channels. We furthermore test the validity of this model by using the active KCNE3 transmembrane domain to functionally rescue a nonconducting, yet assembly and trafficking competent, long QT mutation located in the conserved COOH-terminal region of KCNE1.

PubMed ID: 15572349
PMC ID: PMC2234029
Article link: J Gen Physiol
Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: kcne1 kcne3 kcnq1

Article Images: [+] show captions

	Figure 1. . KCNQ1/KCNE3 channels have a large standing current at negative membrane potentials. (A) Two-electrode voltage clamp recordings from Xenopus oocytes injected with Q1 (left) or Q1/E3 (right). Traces were recorded in KD98 with a 13-s interpulse interval. Dashed line indicates zero current. Inset, “Activation Curve Protocol” of 2-s depolarizations used to elicit currents shown. (B) Voltage–activation curves from Q1 and Q1/E3 channels. Tail currents were measured 3 ms after repolarization, fit to a Boltzmann, and the data normalized such that the upper asymptote was equal to 1. Squares, Q1; circles, Q1/E3. Data were averaged from six oocytes each ± SEM. Scale bars represent 1 μA and 0.5 s.
	Figure 2. . KCNE3 truncation mutants. Solid lines depict which amino acid residues remain in E3 truncation mutants. Numbers across the top denote amino acid residue number. TM, transmembrane domain; N, putative N-linked glycosylation site.
	Figure 3. . KCNE3 NH2-terminal truncation mutants produce standing currents at negative potentials when coexpressed with Q1. (A) TEVC recordings from Xenopus oocytes injected with Q1 and either Δ5-40 (left), Δ10-51 (center), or Δ41-55 (right). Traces were recorded in KD98 with a 13-s interpulse interval. Dashed line indicates zero current. Inset, “Activation Curve Protocol” of 2-s depolarizations used to elicit currents shown. (B) Voltage–activation curves from E3 NH2-terminal truncation mutants. Activation curve data were fit to a Boltzmann, and the data renormalized as described in materials and methods. Squares, Δ5-40; circles, Δ10-51; triangles, Δ41-55. Dotted lines indicate the voltage-independent activation of Q1 and Q1–E3 from the lower asymptotes of Q1 the corresponding Boltzmann fits. Data were averaged from four to six oocytes ± SEM. Scale bars represent 1 μA and 0.5 s.
	Figure 4. . KCNE3 COOH-terminal and combined NH2- and COOH-terminal truncation mutants also exhibit standing currents at negative potentials when coexpressed with Q1. (A) TEVC recordings from Xenopus oocytes injected with Q1 and either Δ84 (left) or Δ10-51/Δ84 (right). Traces were recorded in KD98 with a 13-s interpulse interval. Dashed line indicates zero current. Scale bars represent 1 μA and 0.5 s. Inset, “Activation Curve Protocol” of 2-s depolarizations used to elicit currents shown. (B) Voltage–activation curves from KCNE3 COOH-terminal and double truncation mutants. Data were fit to a Boltzmann, and normalized as described in materials and methods. Squares, Δ84; circles, Δ10-51/Δ84. Dotted lines indicate the voltage-independent activation of Q1 or Q1–E3 (E3). Data were averaged from four oocytes each ± SEM. (C) External K+ concentration was varied and the reversal potential was measured. log[K+]ext is plotted against observed reversal potential. WT and E3 truncation mutants coexpressed with Q1 produced linear fits within error of 53 mV per decade. Data was averaged from four to six oocytes ± SEM.
	Figure 5. . A long QT mutation in the COOH terminus of KCNE1 is masked by the KCNE3 transmembrane domain. Inset, “Activation Curve Protocol” of 2-s depolarizations used to elicit currents shown. Scale bar represents 1 μA and 0.5 s for all recordings. (A) TEVC recording from Xenopus oocytes injected with Q1 and either E1 D76N (left) or E3 D90N (right). (B) Representative TEVC recordings from oocytes coinjected with Q1 and a chimeric partner protein, E1 D76N with the E3 transmembrane (TM) sequence. All oocytes were injected with equal amounts and ratios of Q1 and E1 or E3 RNA, and were recorded in KD98 using a 13-s interpulse interval. Dashed lines indicate zero current.
	Figure 6. . KCNE3 COOH-terminal mutants demonstrate a functional dependence on cRNA injection ratios. (A) Representative two-electrode voltage clamp recordings taken from oocytes injected with varying amounts of E3, between 2.5x and 0.05x of Q1. Currents were recorded in KD98, using a 13-s interpulse interval. The Q1:E3 ratio is labeled across the top. Top row, E3 (square); second row, Δ10-51 (circle); third row, Δ84 (diamond); bottom row, D90N (triangle). Dashed lines indicate zero current. Scale bars represent 1 μA and 0.5 s. Inset, “Activation Curve Protocol” of 2-s depolarizations used to elicit currents shown. (B) Percent current remaining after inhibition with 10 μM chromanol 293B. Oocytes were held at −80 mV and depolarized for 2 s to + 40 mV with a 28-s interpulse interval. Current levels were allowed to stabilize for ≤5 min before 10 μM chromanol 293B was perfused in. Chromanol inhibition typically reached equilibrium within 5 min of wash in. Dashed line indicates the percentage of current remaining in oocytes expressing Q1 alone. Data was averaged from 3–11 oocytes ± SEM.
	Figure 7. . Functional characterization of KCNQ1/KCNE3–HA complexes. (A) Q1 complexes formed with HA-tagged E3 exhibit potassium-dependent inactivation. TEVC recordings of oocytes expressing Q1 and E3-HA were bathed in ND96 (left) and KD98 (right). Both recordings are shown at the same scale, and use a 13-s interpulse interval. Insets, “Activation Curve Protocol” of 2-s depolarizations used to elicit currents shown. (B) Current versus time plotted for potassium concentration changes for the Q1–E3–HA complex shown in A. Oocytes were held at −80 mV, and depolarized for 2 s to + 40 mV, with a 28-s interpulse interval. Squares denote the current measured at +40 mV, triangles at −80 mV 100 ms before the respective capacitive transients. (C) HA-tagged E3 shows altered dose dependence for the COOH-terminal truncation mutant. TEVC measurements of oocytes injected with 0.4x (left) or 0.05x (right) E3–HA relative to Q1. Currents were recorded with 10 mM Rb+ (RD10), and oocytes were held at −40 mV, with an 11.5-s interpulse interval. Top, E3–HA; middle, Δ84–HA; bottom, D90N–HA. Scale bars represent 1 μA and 0.5 s.
	Figure 8. . WT and COOH-terminal mutant HA-tagged KCNE3 peptides are glycosylated and not proteolytically degraded in oocytes. Crude membranes for the immunoblots were prepared 3–5 d after coinjection with Q1 and the indicated E3 construct. (A) Immunoblot of HA-tagged E3 peptides from SDS-solubilized membranes isolated from oocytes injected at a Q1/E3 ratio of 1/0.4. Membranes from three oocytes were loaded in each lane and resolved with a 15% Tris-glycine SDS gel. Molecular weight standards are labeled on the left for both blots. (B) Immunoblots of enzymatically deglycosylated E3 and Δ84 HA-tagged proteins were separated on 16.5% Tris-tricine SDS gels. E3 (four oocytes/lane) and Δ84 (six oocytes/lane) were digested with endoglycosylase Hf (Endo H) or PNGase F. Lane marked (−) represents untreated samples. Loaded in the lane marked 1/0.05 are solubilized membranes from 17 oocytes expressing Q1/Δ84 injected at the lowest Q1/E3 ratio examined. Membranes from uninjected oocytes are denoted and were loaded at the left (4 oocytes) and right (17 oocytes). Mature and immature glycosylation is denoted, as determined by enzymatic deglycosylation. (C) Cell surface expression of HA-tagged E3 proteins was quantitated by single oocyte chemiluminescence. Oocytes were injected with a 1/0.4 Q1/E3 ratio, allowed to incubate for 5 d, and the cell surface–tagged proteins were labeled with an anti-HA antibody followed by a secondary HRP-conjugated antibody. Single oocyte luminescence was quantitated in a luminometer and is reported in relative light units (RLU). Q1 sample is from control oocytes injected with only Q1 RNA. Error bars are standard error measurement from 15–19 oocytes.
	Figure 9. . A bipartite model for modulation of KCNQ1 by KCNE1 and KCNE3. Net diagrams of the transmembrane domains of E1 and E3 are aligned; amino acid residues, denoted as circles, are shaded based on conservation. Black circles, identical amino acids; gray circles, similar amino acids; horizontally striped circles, high-impact amino acids identified by chimeric studies; vertically striped circles, D76 and D90; no fill, nonsimilar amino acids; TM, transmembrane domain.

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