KCNE1 Constrains the Voltage Sensor of Kv7.1 K+ Channels

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

S1 Constrains S4 in the Voltage Sensor Domain of Kv7.1 K+ Channels

Voltage-gated K+ channels comprise a central pore enclosed by four voltage-sensing domains (VSDs). While movement of the S4 helix is known to couple to channel gate opening and closing, the nature of S4 motion is unclear. Here, we substituted S4 residues of Kv7.1 channels by cysteine and recorded whole-cell mutant channel currents in Xenopus oocytes using the two-electrode voltage-clamp technique. In the closed state, disulfide and metal bridges constrain residue S225 (S4) nearby C136 (S1) within the same VSD. In the open state, two neighboring I227 (S4) are constrained at proximity while residue R228 (S4) is confined close to C136 (S1) of an adjacent VSD. Structural modeling predicts that in the closed to open transition, an axial rotation (∼190°) and outward translation of S4 (∼12 Å) is accompanied by VSD rocking. This large sensor motion changes the intra-VSD S1–S4 interaction to an inter-VSD S1–S4 interaction. These constraints provide a ground for cooperative subunit interactions and suggest a key role of the S1 segment in steering S4 motion during Kv7.1 gating

The KCNQ1(KV7.1) C-terminus, a multi-tiered scaffold for subunit assembly and protein interaction

16 páginas, 6 figuras, 4 tablas -- PAGS nros. 5815-5830The Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOH-terminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The first coiled-coil complex is dimeric and undergoes concentration-dependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0Å crystallographic data. Both coiled-coils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation. Previous SectionNext SectionThe KCNQ channels represent a subfamily of voltage-gated K+ (Kv)3 channels, whose members (Kv7.1-5) are expressed in a wide variety of tissues. These channels play a major role in brain and cardiac excitability through the modulation of the cardiac potential waveform, the regulation of action potential generation and propagation, the tuning of neuronal firing patterns, and the modulation of neurotransmitter release (1, 2). Mutations in human KCNQ genes lead to major cardiovascular and neurological disorders, such as the cardiac long QT syndrome or neonatal epilepsy (3).Like all Kv channels, the KCNQ α subunits share a common core structure of six transmembrane segments with a voltage-sensing domain (S1-S4) and a pore domain (S5-S6) (see Fig. 1A), likely to approximate the mammalian Kv1.2 channel α structure described by MacKinnon and co-workers (4). Often, KCNQ α is in complex with the integral membrane auxiliary subunit, known as KCNE1, IsK, or MinK. This protein alters channel properties, such as single channel conductance and activation kinetics while increasing channel density in the plasma membrane (5). Several structural features of the Kv7 α family members distinguish them from the larger Kv channel superfamily. In particular, their primary sequence encodes a large (300-500 residues), intracellular COOH terminus (Fig. 1A). Sequence analysis predicts four helical regions (dubbed A-D) present in all family members (6). Helices C and D are thought to form coiled-coil assemblies. Yeast two-hybrid screens for proteins interacting with the COOH terminus revealed CaM, the ubiquitous Ca2+ sensor protein, as a binding partner (6-8). Moreover, CaM constitutively associates with the channel (6-10). Helices A and B encode the binding site for CaM, and CaM association is required for proper channel assembly and function (6, 7, 9). LQT mutations that disrupt or significantly weaken the CaM interaction result in little or no complex and little channel expression in live cells (8, 11). Thus, CaM acts as an additional auxiliary subunit of the KCNQ channel complex. Additionally, Ca2+-CaM is a Ca2+ sensor for KCNQ1 function, transducing Ca2+ signals to stimulate IKS channels and producing a Ca2+-dependent left shift in the voltage dependence of channel activation. This Ca2+-sensitive IKS-current stimulation could increase the cardiac repolarization reserve, preventing the risk of ventricular arrhythmias. Biochemical and functional studies have identified Kv7 COOH-terminal regions important for channel tetramerization and trafficking (12-16) based on deletion, truncation, and mutagenesis. Little work has directly examined on a protein level the postulated structural modules and their functional correlates. What precisely is the tertiary and quaternary organization of the COOH terminus, and what does it do that distinguishes it from the other Kv channel subfamilies? Using a recombinant bacterial co-expression system (8), we have dissected the KCNQ1 COOH terminus protein complex. Our findings suggest that the KCNQ1 COOH terminus may be divided into two parts, where the membrane proximal half is important for functional expression, folding, and gating of the channel but not oligomerization, whereas the membrane distal half directs folding, oligomerization, partner specificity, and trafficking to the plasma membrane. This COOH terminus is a multifunctional platform, employing relatively simple structural modules to assemble a channel with high specificity in an apparent hierarchical mannerThis work is supported in part by Israel Science Foundation Grant 672/05 and Wolfson Family funds (to B. A.) and by Israel Science Foundation Grant 1201/04 (to J. A. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this factPeer reviewe

Impact of KCNE1 expression on WT Kv7.1 and mutant R228C.

<p>(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 µM 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 µM 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 µM Cu-Phen. Currents were evoked by a train of step depolarization to +30 mV. Similar results have been obtained in 5 other cells.</p

Effect of KCNE1 co-expression with mutant R231W and I235W.

<p>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.</p

Mutations stabilizing KCNQ1 towards the inactivated state.

<p>(A) and (B) Representative current traces of WT and L233W and Q244W, respectively, recorded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001943#pone-0001943-g002" target="_blank">Figure 2 A</a>. (C) Normalized conductance of L233W (n = 13) (black squares), compared to WT (open squares). (D) Percent of macroscopic inactivation of WT, Q244W and L233W (n = 7–20) as measured by the ratio between the sustained and the peak current amplitudes.</p

Effect of KCNE1 co-expression with mutant R237W and R243W.

<p>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.</p

Gating parameters of WT and mutant Kv7.1 channels.

<p>V₅₀ (half activation voltage) and z (equivalent gating charge) were derived from fitting single Boltzmann function; I₆₀ corresponds to the current density measured at +60 mV in pA/pF. ΔG₀ and ΔΔG₀^c were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001943#s4" target="_blank">methods</a>. Data are expressed as mean ± SEM and in parentheses are indicated the number of cells.^*, p<0.05 compared to WT (two-tailed, Student's unpaired t test). NA, not applicable as R231W mutant is a constitutively open K⁺ leak channel.</p

Mutations stabilizing Kv7.1 to the open state.

<p>(A) and (B) Representative current traces of WT and A226W, respectively. From a holding potential of −90 mV, the membrane was stepped for 3 s from −70 mV to +60 mV in 10 mV increments and then repolarized for 1.5 s to −60 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 (n = 6) and V241W (n = 11), respectively, and compared to WT (n = 20) (open squares). The activation curves were fitted using one Boltzmann function. (E) Representative current traces of R231W. Membrane was stepped for 3 s from −140 mV to +60 mV in 20 mV increments and then repolarized for 1.5 s to −60 mV. (F) Current-voltage relations of R231W (n = 8) (black squares) and WT (open squares). Current density (pA/pF) was plotted as a function of step voltages.</p