Abstract
The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the Ca(V)1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553-7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the Ca(V)1.2 channel. Substitution of Ile-781, the Ca(V)1.2 residue corresponding to Ile-745 in Ca(V)1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779-782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of Ca(V)1.2. Moreover there may be a flexible center of helix bending at positions 779-782 of Ca(V)1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.
Highlights
The effects of I781T substitution on CaV1.2 channel activation were investigated after expressing wild-type or mutant CaV1.2 ␣1-subunits together with auxiliary 1a and ␣2-␦1-subunits in tsA-201 cells
In this study we demonstrate a crucial role of a cluster of amino acids (Leu-779, Ala-780, Ile-781, Ala-782) in the pore-forming segment IIS6 in activation and inactivation of the CaV1.2 channel
This finding is related to a recently described channelopathy, where a mutation of the corresponding isoleucine to threonine in CaV1.4 (I745T) causes a severe X-linked retinal disorder [12]
Summary
Mutagenesis—The CaV1.2 ␣1-subunit coding sequence (GenBankTM X15539) in-frame 3Ј to the coding region of a modified green fluorescent protein (GFP) was kindly donated by Dr M. The mutated fragments were cloned into a BamHI-AflII-cassette (nucleotides 1265 and 2689, numbering according to the CaV1.2 ␣1-subunit coding sequence). This cassette was used in creation of C769P, G770P, N771P, Y772P, I773P, L774P, L775P, N776P, V777P, F778P, L779P, A780P, A782P, and V783P. Ionic Current Recordings and Data Acquisition—Barium currents (IBa) through voltage-gated Ca2ϩ channels were recorded at 22–25 °C using the patch clamp technique [19] by means of an Axopatch 200A patch clamp amplifier (Axon Instruments) 36 – 48 h after transfection. Inactivation curves were drawn according to a Boltzmann equation: IBa,inact ϭ Iss ϩ (1 Ϫ Iss)/(1 ϩ exp[(V Ϫ V0.5,inact.)/k)] where V. is membrane potential, V0.5,inact is midpoint voltage, k is the slope factor, and Iss is the fraction of non-inactivating current. Images were acquired using an argon laser (excitation, 488 nm; emission BP505–530 nm emission filter) for the GFP-tagged CaV1.2 ␣1-subunits and a He-Ne laser (excitation, 543 nm; emission filter, LP650 nm) for FM4-64
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