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 ...

The E462R mutation in the fifth position of the AID (alpha1 subunit interaction domain) region in the I-II linker is known to significantly accelerate voltage-dependent inactivation (VDI) kinetics of the L-type CaV1.2 channel, suggesting that the AID region could participate in a hinged-lid type inactivation mechanism in these channels. The recently solved crystal structures of the AID-CaVbeta regions in L-type CaV1.1 and CaV1.2 channels have shown that in addition to E462, positions occupied by Q458, Q459, E461, K465, L468, D469, and T472 in the rabbit CaV1.2 channel could also potentially contribute to a hinged-lid type mechanism. A mutational analysis of these residues shows that Q458A, Q459A, K465N, L468R, D469A, and T472D did not significantly alter VDI gating. In contrast, mutations of the negatively charged E461, E462, and D463 to neutral or positively charged residues increased VDI gating, suggesting that the cluster of negatively charged residues in the N-terminal end of the AID helix could account for the slower VDI kinetics of CaV1.2. A mutational analysis at position 462 (R, K, A, G, D, N, Q) further confirmed that E462R yielded faster VDI kinetics at +10 mV than any other residue with E462R >> E462K approximately E462A > E462N > wild-type approximately E462Q approximately E462G > E462D (from the fastest to the slowest). E462R was also found to increase the VDI gating of the slow CEEE chimera that includes the I-II linker from CaV1.2 into a CaV2.3 background. The fast VDI kinetics of the CaV1.2 E462R and the CEEE + E462R mutants were abolished by the CaVbeta2a subunit and reinstated when using the nonpalmitoylated form of CaVbeta2a C3S + C4S (CaVbeta2a CS), confirming that CaVbeta2a and E462R modulate VDI through a common pathway, albeit in opposite directions. Altogether, these results highlight the unique role of E461, E462, and D463 in the I-II linker in the VDI gating of high-voltage activated CaV1.2 channels.

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu 515 ) to the beginning of S6 (Ala 560 ). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro 527 and Ile 541 was compatible with the presence of a ␣-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions ؊1, 0, ؉1, ؉2 The TRP ion channels form a large class of cationic channels that are related to the product of the Drosophila TRP gene. TRP channels share a similar predicted topology of six transmembrane segments in which the amino acids that link the fifth and sixth transmembrane domains line the pore region (1). According to the recent IUPHAR classification of ion channels (2), the 21 members of the TRP family can be divided by sequence homology into three subfamilies (3, 4) as short (TRPCx), long or melastatin (TRPMx), and osm-9-like or vanilloid-like (TRPVx) channels. The molecular domains that are mostly conserved among TRP channels include part of the S6 segment, ankyrin repeats in the N terminus, and a "TRP domain" in the C terminus (EWKFAR) (5), the latter being absent from TRPV channels. The TRPC and TRPV proteins have 2-4 N-terminal ankyrin domains suggesting that these proteins are coupled to the spectrin-based membrane cytoskeleton.TRP channels vary significantly in their biophysical properties and gating mechanisms. In contrast to other members of the TRP family, TRPV5 and TRPV6 channels show strong inward rectification, exhibit anomalous mole-fraction effect, are activated by low [Ca 2ϩ ] i and inactivated by higher [Ca 2ϩ ] i (6 -8). TRPV5 and TRPV6 are also highly Ca 2ϩ -selective channels with PCa/PNa Ͼ 100. In particular, ECaC-TRPV5 displays a high Ca 2ϩ affinity with a K d of Ϸ2 M (7) that is comparable to the K d of Ϸ1 M for voltage-dependent Ca V channels (9). A single residue in the S5-S6 linker (Asp 542 ) was found to account for the high Ca 2ϩ affinity of ECaC-TRPV5 (7). The absence of the aspartate residue at the equivalent position in the pore region of TRPV1-4 channels might explain, together with the presence of a lysine residue, the Ϸ20-fold lower Ca 2ϩ selectivity of TRPV1-4 channels (10). TRPV5 and TRPV6 channels can also form homo-and heterotetramers suggesting that they are structurally and functionally related (11).There is currently very little structural data available on the pore architecture of Ca 2ϩ -selective TRP channels. It...

The alpha-interacting domain (AID) in the I-II linker of high voltage-activated (HVA) Ca 2؉ channel ␣1 subunits binds with high affinity to Ca V ␤ auxiliary subunits. The recently solved crystal structures of the AIDCa V ␤ complex in Ca V 1.1/1.2 have revealed that this interaction occurs through a set of six mostly invariant residues Glu/Asp 6 (9). Ca V ␤ subunit modulation of LVA T-type (Ca V 3.1-3.3) channels has yet to be reported (10).Ca V ␤ subunits increase peak current density, in part by recruiting the Ca V ␣1 subunit to the plasma membrane (11)(12)(13)(14), by hyperpolarizing the voltage dependence of activation and inactivation, and by increasing the channel opening probability at the single channel level (4, 15). Ca V ␤ subunits increase inactivation kinetics in an isoform-specific manner with Ca V ␤3 Ͼ Ca V ␤1b Ͼ Ca V ␤1a Ͼ Ca V ␤4 Ͼ Ͼ Ca V ␤2a (4). The four known auxiliary Ca V ␤ subunits can potentially associate with any of the six Ca V ␣1 pore-forming subunits of HVA VDCC (Ca V 1.1-1.4, Ca V 2.1-2.3) via the alpha interaction domain (AID) located on the I-II linker of the Ca V ␣1 subunit. The AID motif is absent from LVA T-type (Ca V 3.1-3.3) VDCC for which Ca V ␤ subunit modulation has never been reported. About half of the residues of the AID helix (Gln 1 -Gln 2 -X 3 -Glu 4 -X 5 -X 6 -Leu 7 -X 8 -Gly 9 -Tyr 10 -X 11 -X 12 -Trp 13 -Ile 14 -X 15 -X 16 -X 17 -Glu 18 ) are strictly conserved (in bold) among the six Ca V ␣1 subunits but homology was found to be higher within members of the Ca V 1 and Ca V 2 families. Nonetheless, positions 8, 11, and 15 are occupied by residues that could vary even within the same Ca V family (Table I) (where X can be any residue) might not be the unique determinant of the Ca V ␣1-␤ interaction. In vitro binding experiments have revealed the presence of additional interaction sites of lower affinity on the cytoplasmic loops of Ca V 2.1 (16,17) and on the C-terminal of Ca V 2.3 (18) but the AID appears to be the primary high affinity site of interaction (14, 15).

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

Cell motility is required for physiological processes of wound repair and organogenesis as well as for the pathologic process of tumor invasion; a critical element of cell motility and invasion is the de novo polarized protrusion of pseudopodia via localized actin polymerization (1-5). Pseudopodia formation is associated with cell motility in vitro and tumor cell motility in vivo, and pseudopodial protrusion of surrounding extracellular matrix has been well characterized as an essential element of tumor cell invasion (6 -15).Hepatocyte growth factor (HGF) 1 /scatter factor, secreted by cells of mesodermal and mesenchymal origin, was originally identified for its ability to disrupt epithelial cell-cell interactions and to trigger invasive growth (16 -23). HGF exhibits powerful mitogenic, motogenic, and morphogenic activities on epithelial and endothelial cells expressing the HGF receptor (HGF-R), also known as the met proto-oncogene (17, 24), whose two end results are the modification of the actin cytoskeleton and the disruption of epithelial cell-cell adhesions (16). HGF-R activation promotes receptor auto-phosphorylation on tyrosine residues and activation of downstream signaling events including the ras (25), phosphatidylinositol 3-kinase (26, 27) phospholipase C-␥ (28), and mitogen-activated protein kinase (29) related pathways. Deregulated control of the invasive-growth phenotype by oncogenically activated Met confers invasive and metastatic properties to cancer cells (18,22). Breast (30), ovarian (31), prostate (32), gastric (33), thyroid (34), hepatocellular (35), and renal (36) carcinomas as well as osteosarcoma (37, 38) and myeloma (39) are indeed associated with HGF-R overexpression or increased HGF-R tyrosine phosphorylation. Notably, protein overexpression was found to be associated with amplification of the met gene in only a few primary carcinomas, but in a significant proportion of the metastases examined (18,33,38). Targeting of a constitutively active Tpr-Met to the plasma membrane via a c-Src myristoylation signal induces enhanced cellular transformation and the formation of cellular protrusions in MDCK cells (40). However, whether the acquisition of a motile and metastatic phenotype by tumor cells due to HGF-R activation is indirectly due to disruption of epithelial cell-cell contacts and induction of an epithelial-mesenchymal transformation or whether autocrine HGF-R activation specifically induces cell motility and, more particularly, pseudopodial protrusion, has yet to be directly demonstrated.To determine the molecular basis for the acquisition of motile and invasive properties following transformation of polarized epithelial cells, we have established a model system based on

The transient receptor potential type V5 (TRPV5) channel is a six-transmembrane domain ion channel that is highly selective to Ca 2ϩ . To study the topology of the selectivity filter using the substituted cysteine accessibility method (SCAM), cysteine mutants at positions 541-547 were studied as heterotetramers using dimeric constructs that couple the control channel in tandem with a cysteine-bearing subunit. Whole cell currents of dimeric constructs D542C, G543C, P544C, A545C, and Y547C were rapidly inhibited by positively charged 2-(trimethyl ammonium)methyl methane thiosulfonate bromide (MTSMT), 2-(aminoethyl)methane thiosulfonate bromide (MTSEA), and 2-(trimethyl ammonium)ethyl methane thiosulfonate bromide (MTSET) reagents, whereas D542C, P544C, and A545C were inhibited only by negatively charged sodium 2-(sulfonatoethyl)methane thiosulfonate (MTSES). In contrast, the I541C dimer remained insensitive to positive and negative reagents. However, I541C/D542G and I541C/D542N dimeric constructs were rapidly (Ͻ30 s) and strongly inhibited by positively and negatively charged methane thiosulfonate reagents, suggesting that removing two of the four carboxylate residues at position 542 disrupts a constriction point in the selectivity filter. Taken together, these results establish that the side chains of contiguous amino acids in the selectivity filter of TRPV5 are rapidly accessible from the external medium, in contrast to the three-dimensional structure of the selectivity filter in K ϩ channels, where main chain carbonyls were shown to project toward a narrow permeation pathway. The I541C data further suggest that the selectivity filter of the TRPV5 channel espouses a specific conformation that restrains accessibility in the presence of four carboxylate residues at position 542. calcium; kidney; transport; cysteine; site-directed mutagenesis; electrophysiology; methane thiosulfonate reagents; three-dimensional homology modeling; ion channel; transient receptor potential TRANSIENT RECEPTOR POTENTIAL (TRP) type V (TRPV) channels belong to the six-transmembrane (6-TM) family of ion channels with both NH 2 -and COOH-termini located intracellularly (18). TRPV5 (ECaC1 and CaT2) and TRPV6 (ECaC2, CaT1, and CaT-L) play key roles in renal Ca 2ϩ reabsorption and intestinal Ca 2ϩ absorption, respectively. They both form a distinctive subgroup within the TRP family, as they show strong inward rectification, exhibit an anomalous mole-fraction effect, are activated by low intracellular (9) and is proposed to form the main binding site for divalent cations within the selectivity filter (9, 14). Assuming that Asp 542 controls the pore diameter, the diameter of the selectivity filter has been estimated to vary from 5.4 Å (23) for TRPV6 to 8.5 Å (25) for TRPV5 at physiological pH. Altogether, these observations suppose that the negatively charged carboxylate groups of Asp 542 /Asp 541 project toward the pore lumen and that the selectivity filter of TRPV5/6 is wider than in K ϩ channels. This structural arrangement would contrast w...

Rem2 is a small GTP-binding protein of the RGK family. It is targeted to the cell membrane where it interacts with the beta subunit of calcium channels and abolishes or reduces endogenous or exogenous calcium currents, and also has known interactions with calmodulin and 14-3-3. Rem2 is unique in the RGK family, being found predominantly in the brain and upregulated in response to stimulation. Knockdown of Rem2 in neuronal cultures results in fewer glutamatergic synapses. We have found that fluorescent-labeled Rem2 changes its subcellular localization in neurons from a diffuse to a punctuate distribution after neuronal stimulation or after activation of NMDA receptors. This rearrangement is calcium dependent and involves the C-terminal 30 residues, suggesting the presence of a self-association domain as well as an autoinhibitory domain that keeps Rem2 diffusely distributed until stimulation. A calmodulin-binding deficient mutant shows very little rearrangement upon stimulation, supporting a role for calcium in this phenomenon.