Pantazis A et al. (2010), Relative motion of transmembrane segments S0 an...

XB-ART-42957

J Gen Physiol 2010 Dec 01;1366:645-57. doi: 10.1085/jgp.201010503.

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Relative motion of transmembrane segments S0 and S4 during voltage sensor activation in the human BK(Ca) channel.

Pantazis A , Kohanteb AP , Olcese R .

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Large-conductance voltage- and Ca(2+)-activated K(+) (BK(Ca)) channel α subunits possess a unique transmembrane helix referred to as S0 at their N terminus, which is absent in other members of the voltage-gated channel superfamily. Recently, S0 was found to pack close to transmembrane segments S3 and S4, which are important components of the BK(Ca) voltage-sensing apparatus. To assess the role of S0 in voltage sensitivity, we optically tracked protein conformational rearrangements from its extracellular flank by site-specific labeling with an environment-sensitive fluorophore, tetramethylrhodamine maleimide (TMRM). The structural transitions resolved from the S0 region exhibited voltage dependence similar to that of charge-bearing transmembrane domains S2 and S4. The molecular determinant of the fluorescence changes was identified in W203 at the extracellular tip of S4: at hyperpolarized potential, W203 quenches the fluorescence of TMRM labeling positions at the N-terminal flank of S0. We provide evidence that upon depolarization, W203 (in S4) moves away from the extracellular region of S0, lifting its quenching effect on TMRM fluorescence. We suggest that S0 acts as a pivot component against which the voltage-sensitive S4 moves upon depolarization to facilitate channel activation.

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Species referenced: Xenopus
Genes referenced: acta4 kcna2 tbx2

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	Figure 1. Side and top views of the putative structure of the BKCa channel. Only two out of four Î± subunits are shown for clarity. Each Î± subunit consists of seven transmembrane segments (S0âS6) and a large intracellular ligand-binding domain. Segments S0âS4 comprise the VSD, whereas segments S5 and S6 from all four subunits contribute to the central, K+-selective pore (K+ ions occupying the pore are shown as purple spheres). Each subunit also contributes an intracellular RCK1/RCK2 heterodimer, which assembles into the hetero-octameric gating ring superstructure. The structure shown for domains S1âS6 is from the atomic structure of the KV1.2-2.1 chimera (Protein Data Bank accession no. 2R9R) (Long et al., 2007); S0 was modeled as an ideal Î± helix. Note its close association with voltage-sensing segments S3 and S4, as recently suggested by Liu et al. (2010). The intracellular domain structures (available from Protein Data Bank accession no. 3NAF) (Wu et al., 2010) were manually docked on the 2R9R structure.
	Figure 2. Voltage-dependent conformational rearrangements reported from the N-terminal flank of S0. (A) A unique cysteine was substituted at position 17 at the putative N-terminal flank of S0 in a BKCa channel Î± subunit and covalently labeled with the fluorophore TMRM to resolve conformational rearrangements from this region. (B) Voltage pulses and characteristic evoked K+ currents from BKCa channels labeled with TMRM at position 17. (C) TMRM fluorescence traces recorded during the voltage pulses in B. (D) Normalized K+ conductance (G; black circles) and ÎF/F (red squares) plotted against membrane potential and fitted with Boltzmann distributions (black and red curves, respectively). Boltzmann parameters are listed in Table I. Error bars represent SEM. (EâH) As in AâD, respectively, for BKCa channels labeled with TMRM at position 18. (IâL) As in AâD, respectively, for BKCa channels labeled with TMRM at position 19.
	Figure 3. Neutralization of R20 alters channel activation but does not prevent conformational rearrangements reported from the S0 region. (A) Putative topology of the BKCa channel Î± subunit voltage sensor domain. A unique cysteine was substituted at position 19 at the putative N-terminal flank of S0, bearing mutation R20A and covalently labeled with the fluorophore TMRM to resolve conformational rearrangements from this region. (B) Voltage pulses and characteristic evoked K+ currents from R20A BKCa channels labeled with TMRM at position 19. (C) TMRM fluorescence traces recorded during the voltage pulses in B. (D) Normalized K+ conductance from channels with mutation R20A (black circles) and without (gray triangles) plotted against membrane potential and fitted with Boltzmann distributions (black and gray curves, respectively). Boltzmann parameters are listed in Table I. Error bars represent SEM. (E) Normalized TMRM fluorescence from channels with mutation R20A (red squares) and without (pink diamonds), plotted against membrane potential and fitted with Boltzmann distributions (red and pink curves, respectively). Boltzmann parameters are listed in Table I. Error bars represent SEM. (FâJ) As in AâE, respectively, for BKCa channels labeled with TMRM at position 145 at the extracellular tip of S2. (KâO) As in AâE, respectively, for BKCa channels labeled with TMRM at position 202 at the short extracellular linker between segments S3 and S4. Mutation W203V was included to enhance ÎF/F signals from position 202 (Savalli et al., 2006). Mutation R20A impaired the activation of all TMRM-labeled BKCa channels investigated, as it shifted the midpoint of activation (Vhalf) toward depolarized potentials by â30 mV (see Table I).
	Figure 4. TMRM at position 20 tracks two distinct voltage-dependent processes. (A) A unique cysteine was substituted at position 20 at the putative N-terminal flank of S0 in a BKCa channel Î± subunit and covalently labeled with the fluorophore TMRM to resolve conformational rearrangements from this region. (B) Voltage pulses and characteristic evoked K+ currents from BKCa channels labeled with TMRM at position 20. (C) TMRM fluorescence traces recorded during the voltage pulses in B. Note that, contrary to ÎF/F signals observed from other positions in BKCa, fluorescence recordings to 20 or 80 mV exhibit transient deflections at the onset and termination of the test pulse. (D) Normalized K+ conductance (black circles) fit with a Boltzmann distribution (black curves). Boltzmann parameters are listed in Table I. Error bars represent SEM. (E) Normalized steady-state fluorescence. The biphasic relationship implies that two processes with opposite effects on fluorescence intensity (quenching/dequenching) and steady-state voltage dependence influence TMRM fluorescence when labeling position 20. (F) Magnification of the fluorescence trace for a 20-mV depolarization from C. In this scale, the fluorescence transients (events marked 1 and 3) are more easily observed.
	Figure 5. A large component of the ÎF reported from the S0 extracellular flank is abolished by the substitution of W203 at the extracellular tip of S4. (A) Putative topology of the BKCa channel Î± subunit voltage sensor domain. A unique cysteine was substituted at position 18 at the putative N-terminal flank of S0 to resolve conformational rearrangements from this region. W203 is also indicated in green. (B) TMRM fluorescence traces recorded during depolarization to 40 mV from wt channels labeled at position 18 (red trace, as in Fig. 2 G) and channels bearing mutation W203V (blue trace). Note the dramatically diminished ÎF/F signal amplitude. (C) Normalized TMRM fluorescence from channels with mutation W203V (blue diamonds) and without (red squares), plotted against membrane potential and fitted with Boltzmann distributions (blue and red curves, respectively). Boltzmann parameters are listed in Table I. Error bars represent SEM. (DâF) As in AâC, respectively, for wt and W203V channels labeled at position 20. In this position, the fluorophore is apparently influenced by two voltage-dependent processes with opposite effects on its fluorescence intensity (Fig. 4). Mutation W203V diminishes the voltage-dependent unquenching process, but it does not apparently affect the residual voltage-dependent quenching process (E). (G) Mean fitted ÎFmax/F signal, normalized for fitted maximum conductance to normalize for channel expression, plotted against labeled position, for wt and W203V channels. Mutation W203V diminishes the ÎF/F observed from positions 17â19 by â90%. At position 20, mutation W203V removes the unquenching process (positive ÎF/F) to reveal a residual negative ÎF/F signal. Error bars represent SEM.
	Figure 6. A tryptophan derivative can quench TMRM in solution. (A) Molecular structure of NATA, an uncharged l-tryptophan derivative that resembles the structure of nonterminal Trp in proteins. (B) The molecular structure of l-tryptophan. (C) Fluorescence spectra of 1 ÂµM TMRM in external solution under increasing [NATA]. (D) Stern-Volmer plot of peak TMRM fluorescence (573 nm) quenched by NATA. The Stern-Volmer constant (KSV) is 41.3 Mâ1.
	Figure 7. A proposed relative, activation-dependent motion between S0 and S4. (A; left) A hypothetical model of the BKCa voltage sensor at rest, showing helices S0, S3, and S4 arranged according to the most recent information from disulfide cross-linking efficiency (Liu et al., 2010). S3 (blue) and S4 (red) helices are modeled by homology with the KV1.2-2.1 chimera structure (Protein Data Base accession no. 2R9R) (Long et al., 2007). The two helices are joined by a short helixâloopâhelix structure as previously inferred by bimane fluorescence (Semenova et al., 2009). The S0 helix (olive) is modeled as an ideal Î± helix, whereas its N-terminal flank is shown as a disordered coil (black). A substituted cysteine at position 18 (yellow) is bound by a TMRM molecule (black), which is in close proximity to W203 at the extracellular tip of S4 (green). In this state, TMRM fluorescence is quenched by W203. Voltage-sensing residues D186 (in S3) and R213 (in S4) (Ma et al., 2006) are also shown. (Right) Membrane depolarization induces R213 to move outwards and D186 inwards, causing activation of the voltage sensor domain. This results in a relative motion between the voltage-sensing S3/S4 and S0, increasing the distance between W203 and the fluorophore. As a result, the quenching effect is lifted, producing a large, positive ÎF/F signal. (B) As in A, for channels with mutation W203V. Because of the absence of W203, TMRM is at a bright state when the VSD is at rest and only exhibits a small deflection during voltage sensor activation. (C) Characteristic TMRM fluorescence traces for wt and W203V channels (red and blue, respectively) labeled at position 18. The traces are superimposed at their fluorescence level when the membrane is depolarized. Fluorescence from wt channels at â160 mV (resting VSD state) is quenched because of the intimate association of the fluorophore with W203 (A). In contrast, the fluorophore is not quenched in W203V channels at rest (B) and emits more fluorescence. TMRM fluorescence in both wt and W203V channels increases upon depolarization after a large or small fluorescence deflection, respectively.

References [+] :

Armstrong, Voltage-gated K channels. 2003, Pubmed