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 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
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...
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