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).