Yolaine Dodier

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

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) CaV channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in CaV1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly432 and Gly436 in CaV1.2) to form a triglycine motif unique to HVA CaV channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca2+ channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In CaV1.2, α-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 CaV1.2 wild type > G770A > G422A ≅ G436A >> G432A (from the fastest to the slowest). Mutations at position Gly432 produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly436 to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX3G residues blunted Ca2+-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In CaV2.3, mutation of the distal glycine Gly352 impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX3G motif in the voltage-dependent activation and inactivation gating of HVA CaV channels with the distal glycine residue being mostly involved in the inactivation gating.

Autocrine Activation of the Hepatocyte Growth Factor Receptor/Met Tyrosine Kinase Induces Tumor Cell Motility by Regulating Pseudopodial Protrusion

The multiple β-actin rich pseudopodial protrusions of the invasive variant of Moloney sarcoma virus (MSV)-transformed epithelial MDCK cells (MSV-MDCK-INV) are strongly labeled for phosphotyrosine. Increased tyrosine phosphorylation among a number of proteins was detected in MSV-MDCK-INV cells relative to untransformed and MSV-transformed MDCK cells, especially for the hepatocyte growth factor receptor (HGF-R), otherwise known as c-met proto-oncogene. Cell surface expression of HGF-R was similar in the three cell lines, indicating that HGF-R is constitutively phosphorylated in MSV-MDCK-INV cells. Both the tyrosine kinase inhibitor herbimycin A and the HGFα antibody abolished HGF-R phosphorylation, induced retraction of pseudopodial protrusions, and promoted the establishment of cell-cell contacts as well as the apparition of numerous stabilizing stress fibers in MSV-MDCK-INV cells. Furthermore, anti-HGFα antibody abolished cell motility among MSV-MDCK-INV cells. Conditioned medium from MSV-MDCK-INV cells induced MDCK cell scattering, indicating that HGF is secreted by MSV-MDCK-INV cells. HGF titration followed by a subsequent washout of the antibodies led to renewed pseudopodial protrusion and cellular movement. We therefore show that activation of the tyrosine kinase activity of HGF-R/Met via an autocrine HGF loop is directly responsible for pseudopodial protrusion, thereby explaining the motile and invasive potential of this model epithelium-derived tumor cell line.

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

The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in CaV1 and CaV2 families. Herein we show that glycine substitution of the VAVIM motif in CaV2.3 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 (E0.5,act). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of ≈-20 mV in their E0.5,act indicating that the relative stability of the channel closed state was decreased in these mutants. Glycine scan performed with Val 349 in IS6, Ile701 in IIS6, and Leu1420 in IIIS6 at positions predicted to face Val1720 in IVS6 also produced slow inactivating currents with hyperpolarizing shifts in the activation and inactivation potentials, again pointing out a decrease in the stability of the channel closed state. Mutations to other hydrophobic residues at these positions nearly restored the channel gating. Altogether these data indicate that residues at positions equivalent to 1720 exert a critical control upon the relative stability of the channel closed and open states and more specifically, that hydrophobic residues at these positions promote the channel closed state. We discuss a three-dimensional homology model of CaV2.3 based upon Kv1.2 where hydrophobic residues at positions facing Val1720 in IS6, IIS6, and IIIS6 play a critical role in stabilizing the closed state in CaV2.3.