Functional Architecture of the Inner Pore of a Voltage-gated Ca2+ Channel

Zhen, Xiao-guang; Xie, Cheng; Fitzmaurice, Aileen; Schoonover, Carl E.; Orenstein, Eleza T.; Yang, Jian

doi:10.1085/jgp.200509292

Cited by 42 publications

(74 citation statements)

References 42 publications

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“…C, the ⌬ of the mid-points of inactivation E 0.5,inact (striped columns) and activation E 0.5,act (gray columns) were calculated as described before in the presence of Ca V ␤3 such that a ⌬ value ϭ 0 indicates no difference from the wild-type channel (see supplementary Table S2 for actual values). The dotted lines drawn at Ϯ 5 mV around 0 mV indicates a value significantly different form the wild-type channel by p Ͻ 0.01. the selectivity filter of Ca V 1.2 (41,42) and recently substantiated by the SCAM study of Ca V 2.1 (43,44).…”

Section: Resultsmentioning

confidence: 66%

The Role of Distal S6 Hydrophobic Residues in the Voltage-dependent Gating of CaV2.3 Channels

Raybaud

Baspinar

Dionne

et al. 2007

Journal of Biological Chemistry

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The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in Ca V 1 and Ca V 2 families. Herein we show that glycine substitution of the VAVIM motif in Ca V 2.3 produced whole cell currents with inactivation kinetics that were either slower (A1719G ≈ V1720G), similar (V1718G), or faster (I1721G ≈ M1722G) than the wild-type channel. The fast kinetics of I1721G were observed with a ≈؉10 mV shift in its voltage dependence of activation (E 0.5,act ). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of ≈؊20 mV in their E 0.5,act indicating that the relative stability of the channel closed state was decreased in these mutants. Voltage-dependent Ca 2ϩ channels (VDCC) 3 are membrane proteins that play a key role in promoting Ca 2ϩ influx in response to membrane depolarization in excitable cells. VDCCs arise from the multimerization of distinct subunits: Ca V ␣1, Ca V ␤, and Ca V ␣2␦, and sometimes Ca V ␥ (1). To this date, molecular cloning has identified the primary structures for 10 distinct calcium channel Ca V ␣ 1 subunits (2-8) that are classified into three main subfamilies according to their gating properties (Ca V 1, Ca V 2, and Ca V 3). The Ca V ␣1 subunit is the main pore-forming subunit that carries the channel activation gating among other functions. The Ca V ␣1 subunits of VDCCs are evolutionarily related to the ␣ subunit of Kv channels with a single polypeptidic chain carrying four domains of six transmembrane segments (S1-S6) (9). Although the overall identity at the primary sequence level is very low between Ca V and Kv channels, it goes up to 10 -25% when comparing the S6 transmembrane segments. As in Kv channels, the S6 transmembrane segments of Ca V ␣1 are believed to line the channel pore and form the channel inner vestibule. It was inferred from the three-dimensional structures of KcsA, MthK, KvAP, KirBac, and Kv1.2 channels that the M2/S6 transmembrane segments include the activation gate that controls channel opening (10 -14). In the Shaker K ϩ channels, the residue hydrophobicity in this region could alter the channel closed-open equilibrium (15,16).To study the functional importance of S6 residues in the gating of Ca V 2.3, we searched for conserved motifs of hydrophobic residues. The VAVIM motif is conserved in the S6 transmembrane segment of domain IV (IVS6) of high voltageactivated Ca V 1 and Ca V 2 families (Fig. 1A). Numerous algorithms align the PVPVIV activation locus in Shaker Kv channels with the FVAVIM (50% identity) (Fig. 1B) suggesting that this locus could play a role in the activation gating of HVA VDCCs. Precious clues regarding the role of the VAVIM motif in channel function came from genetic diseases. Mutation of the conserved Ile to Leu in Ca V 2

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Section: Resultsmentioning

confidence: 66%

The Role of Distal S6 Hydrophobic Residues in the Voltage-dependent Gating of CaV2.3 Channels

Raybaud

Baspinar

Dionne

et al. 2007

Journal of Biological Chemistry

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“…This structural arrangement predicts that the second glycine in the GX 9 GX 3 G motif could remain inaccessible from the water-based pore in both Ca V 1.2 and Ca V 2.3 channels. SCAM studies of the HVA Ca V 2.1 channel support the inaccessibility of this residue from the cytoplasmic medium (77). Although the distal Gly 436 and Gly 352 are also predicted to lie away from the inner pore, their location at the S6/cytoplasm interface could facilitate their interactions with the cytoplasmic environment.…”

Section: Discussionmentioning

confidence: 97%

The Role of the GX9GX3G Motif in the Gating of High Voltage-activated Ca2+ Channels

Raybaud

Dodier

Bissonnette

et al. 2006

Journal of Biological Chemistry

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The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) Ca V channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca V 1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly 432 and Gly 436 in Ca V 1.2) to form a triglycine motif unique to HVA Ca V channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca 2؉ channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca V 1.2, ␣-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with Ca V 1.2 wild type > G770A > G422A Х G436A Ͼ Ͼ G432A (from the fastest to the slowest). Mutations at position Gly 432 produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly 436 to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX 3 G residues blunted Ca 2؉ -dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca V 2.3, mutation of the distal glycine Gly 352 impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX 3 G motif in the voltage-dependent activation and inactivation gating of HVA Ca V channels with the distal glycine residue being mostly involved in the inactivation gating.Voltage-dependent calcium channels are membrane-bound proteins that form large aqueous pores for the selective diffusion of Ca 2ϩ ions across the plasma membrane (1, 2). Native Ca 2ϩ channels are composed of the pore-forming Ca V ␣1, the disulfur-linked dimer Ca V ␣2␦, the intracellular Ca V ␤ subunits (␤1-␤4), and in some cases the Ca V ␥ subunit (3). To date, molecular cloning has identified the primary structures for 10 distinct calcium channel Ca V ␣ 1 subunits (1, 4 -9) that are classified into three main subfamilies according to their gating properties (Ca V 1, Ca V 2, and Ca V 3). Whereas all voltage-gated Ca 2ϩ channel ␣1 subunits activate and inactivate in response to membrane depolarization, the high voltage-activated (HVA) 2 Ca V 1 and Ca V 2 ␣1 subunits operate at markedly more positive membrane potentials than low voltage-activated Ca V 3 channel ␣1 subunits.In the absence of a crystal structure for these proteins, details regarding the structural determinants of the channel inner pore as well as the molecular mechanism underlying the activation of Ca V ␣1 subunits remain sketchy. Structural studies have revealed that the architecture of the ion-selective pore is conserved in the homologous ␣ subunit of different K ϩ channels (10 -15) with the ...

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“…Residue substitution experiments associated with crystal structure analysis extrapolated from K + channels have demonstrated that S6 segments play a central role in voltage-dependent channel gating (Zhou et al, 2001;Hohaus et al, 2005;Long et al, 2005;Zhen et al, 2005;Beyl et al, 2007;Long et al, 2007;Kudrnac et al, 2009). In particular, glycine residues of the GX 9 GX 3 G motif are involved in activation and inactivation mechanisms: mutations of these residues lead to a shift of the half-activation potential and a decrease of inactivation speed (Splawski et al, 2004;Splawski et al, 2005;Raybaud et al, 2006;Barrett and Tsien, 2008;Cens et al, 2008;Depil et al, 2011;Yazawa et al, 2011).…”

Section: Research Articlementioning

confidence: 99%

Hyperactivation of L-type voltage-gated Ca2+ channels in C. elegans striated muscle can result from point mutations in the IS6 or the IIIS4 segment of the α1 subunit.

Lainé

Ségor

Zhan

et al. 2014

Journal of Experimental Biology

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Several human diseases, including hypokalemic periodic paralysis and Timothy syndrome, are caused by mutations in voltage-gated calcium channels. The effects of these mutations are not always well understood, partially because of difficulties in expressing these channels in heterologous systems. The use of Caenorhabditis elegans could be an alternative approach to determine the effects of mutations on voltage-gated calcium channel function because all the main types of voltage-gated calcium channels are found in C. elegans, a large panel of mutations already exists and efficient genetic tools are available to engineer customized mutations in any gene. In this study, we characterize the effects of two gain-of-function mutations in egl-19, which encodes the L-type calcium channel α 1 subunit. One of these mutations, ad695, leads to the replacement of a hydrophobic residue in the IIIS4 segment. The other mutation, n2368, changes a conserved glycine of IS6 segment; this mutation has been identified in patients with Timothy syndrome. We show that both egl-19 (gain-of-function) mutants have defects in locomotion and morphology that are linked to higher muscle tone. Using in situ electrophysiological approaches in striated muscle cells, we provide evidence that this high muscle tone is due to a shift of the voltage dependency towards negative potentials, associated with a decrease of the inactivation rate of the L-type Ca 2+ current. Moreover, we show that the maximal conductance of the Ca 2+ current is decreased in the strongest mutant egl-19(n2368), and that this decrease is correlated with a mislocalization of the channel.

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Functional Architecture of the Inner Pore of a Voltage-gated Ca2+ Channel

Cited by 42 publications

References 42 publications

The Role of Distal S6 Hydrophobic Residues in the Voltage-dependent Gating of CaV2.3 Channels

The Role of Distal S6 Hydrophobic Residues in the Voltage-dependent Gating of CaV2.3 Channels

The Role of the GX9GX3G Motif in the Gating of High Voltage-activated Ca2+ Channels

Hyperactivation of L-type voltage-gated Ca2+ channels in C. elegans striated muscle can result from point mutations in the IS6 or the IIIS4 segment of the α1 subunit.

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