Negatively Charged Residues in the N-terminal of the AID Helix Confer Slow Voltage Dependent Inactivation Gating to CaV1.2

Abstract

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

“…Point mutations were produced with the QuikChange XL mutagenesis kit (Stratagene, La Jolla, CA) using 39-bp primers as detailed before (34). Ca V 1.2 mutations were performed by cassette cloning using the naturally occurring SacI (956) site and the XhoI site that was engineered at nucleotide 1530 in the I-II linker of Ca V 1.2 (42 residues before IIS1) (34 -36).…”

“…Functional Expression of Wild-type and Mutant ChannelsOocytes were obtained from female Xenopus laevis clawed frog (Nasco, Fort Atkinson, WI) as described previously (34,37). Individual oocytes free of follicular cells were obtained after a 30 -40-min incubation in a calcium-free solution: 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 5 mM Hepes, pH 7.6, contain- Membrane expression of nonfunctional mutants G422P, G432P, and G422A/ G432A was assessed from Western blots.…”

“…Electrophysiological Recordings in Oocytes-Wild-type and mutant channels were screened at room temperature for macroscopic Ba 2ϩ currents 2-6 days after RNA injection using a two-electrode voltage clamp amplifier (OC-725C; Warner Instruments) as described earlier (34,35,37). Voltage and current electrodes were filled with 3 M KCl, 1 mM EGTA, 10 mM HEPES (pH 7.4).…”

“…In a few experiments, the voltage dependence of inactivation (isochronal inactivation) were obtained from normalized currents measured at 0 or ϩ10 mV after a series of 5-s prepulses that varied from Ϫ100 to ϩ30 mV at a frequency of 0.02 Hz (34). For the isochronal inactivation figures, data points represent the mean of n Ն 4 and were fitted to a Boltzmann equation as described elsewhere (37).…”

“…In particular, it has been shown that negatively charged residues at the fifth position of the ␣ interacting domain in the I-II linker significantly decreased the VDI kinetics and voltage dependence of Ca V 2.3 (35). Furthermore, the negatively charged EED cluster (Glu 461 , Glu 462 , Asp 463 ) could account for the typically slow VDI kinetics of Ca V 1.2 (34). Introducing a positive residue at the fifth position in the L-type Ca V 1.2 with E462R accelerated significantly VDI kinetics (34,35,58).…”