Physicochemical properties of pore residues predict activation gating of CaV1.2: A correlation mutation analysis

Abstract

Single point mutations in pore-forming S6 segments of calcium channels may transform a high-voltage-activated into a low-voltage-activated channel, and resulting disturbances in calcium entry may cause channelopathies (Hemara-Wahanui et al., Proc Natl Acad Sci U S A 102(21):7553–7558, 16). Here we ask the question how physicochemical properties of amino acid residues in gating-sensitive positions on S6 segments determine the threshold of channel activation of CaV1.2. Leucine in segment IS6 (L434) and a newly identified activation determinant in segment IIIS6 (G1193) were mutated to a variety of amino acids. The induced leftward shifts of the activation curves and decelerated current activation and deactivation suggest a destabilization of the closed and a stabilisation of the open channel state by most mutations. A selection of 17 physicochemical parameters (descriptors) was calculated for these residues and examined for correlation with the shifts of the midpoints of the activation curve (ΔVact). ΔVact correlated with local side-chain flexibility in position L434 (IS6), with the polar accessible surface area of the side chain in position G1193 (IIIS6) and with hydrophobicity in position I781 (IIS6). Combined descriptor analysis for positions I781 and G1193 revealed that additional amino acid properties may contribute to conformational changes during the gating process. The identified physicochemical properties in the analysed gating-sensitive positions (accessible surface area, side-chain flexibility, and hydrophobicity) predict the shifts of the activation curves of CaV1.2.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-010-0885-2) contains supplementary material, which is available to authorized users.

CaV3.1 channel pore pseudo-symmetry revealed by selectivity filter mutations in their domains I/II

“…1, squares), slowing its inactivation kinetics and having mild effects on the channel’s deactivation kinetics (Table 1). These results are consistent with previous observations where a left shift of the activation curve is associated with slow deactivation kinetics in a LVA mutant L-type Ca 2+ channel [20]. Interestingly, a recent report pointed out the relevance of the glutamate pore residue of domain II of CaV1.2 on Ca 2+ -dependent inactivation (CDI) of HVA Ca 2+ channels [21], where the selectivity filter forms a CDI regulatory gate, suggesting a selectivity filter contribution to the inactivation paradigm of HVA Ca 2+ channels.…”

“…Indeed, LVA Ca 2+ channels contain a large conserved external loop (residues 211-336 for Ca V 3.1 channel) between the S5 transmembrane segment and the P pore loop of domain I [17], which is different in length with respect to the other three external loops and supports the pseudo-symmetry in LVA channels. At least two different reports have explored the role of pore-lining S6 segment residues in Ca V 1.2 and Ca V 3.2 channels [20,23]. The distal S6 transmembrane segment seems to be especially important for the voltage sensitivity of activation and the activation and deactivation kinetics of Ca V 1.2 channel; substitution of S6 pore region residues of domain I (IS6 L(434), domain II (IIS6; I781) or domain III (IIIS6; G1193) transformed the Ca V 1.2 from a HVA into a LVA Ca 2+ channel, probably for altering backbone-backbone helix interactions in a hydrophobic environment (reviewed in [20]).…”

Timothy Mutation Disrupts the Link between Activation and Inactivation in CaV1.2 Protein

“…A proline mutation of Ala-780 ( shaded ) was excluded from correlation analysis as this residue is likely to disturb the link between voltage sensor and the channel pore (9, 24). …”

Calcium channel gating

“…b Amino acid substitutions in position I781 (corresponding to the channelopathy mutation I745T in CaV1.4, [44]) destabilize the closed conformation and stabilize the open conformation of CaV1.2. In other words, changes in hydrophobicity in position I781 predict the shifts of the activation curve (see also Beyl et al [14] for the role of other amino acid descriptors). Figure from Pflügers Archiv – European Journal of Physiology .…”

“…However, hydrophobic interactions [58] are not the only interactions that contribute to closed state stability. Combined descriptor analysis addressing several amino acid properties simultaneously can substantially improve the correlation indicating that a combination of different amino acid properties contributes to the stability of open or closed conformations (see [14]).…”