Michaela Kudrnac

Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder

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 CaV1.4 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 CaV1.2 channel. Substitution of Ile-781, the CaV1.2 residue corresponding to Ile-745 in CaV1.4, 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 CaV1.2. Moreover there may be a flexible center of helix bending at positions 779–782 of CaV1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.

Probing the Architecture of an L-type Calcium Channel with a Charged Phenylalkylamine EVIDENCE FOR A WIDELY OPEN PORE AND DRUG TRAPPING

Voltage-gated calcium channels are in a closed conformation at rest and open temporarily when the membrane is depolarized. To gain insight into the molecular architecture of Cav1.2, we probed the closed and open conformations with the charged phenylalkylamine (-)devapamil ((-)qD888). To elucidate the access pathway of (-)D888 to its binding pocket from the intracellular side, we used mutations replacing a highly conserved Ile-781 by threonine/proline in the pore-lining segment IIS6 of Cav1.2 (1). The shifted channel gating of these mutants (by 30–40 mV in the hyperpolarizing direction) enabled us to evoke currents with identical kinetics at different potentials and thus investigate the effect of the membrane potentials on the drug access per se. We show here that under these conditions the development of channel block by (-)qD888 is not affected by the transmembrane voltage. Recovery from block at rest was, however, accelerated at more hyperpolarized voltages. These findings support the conclusion that Cav1.2 must be opening widely to enable free access of the charged (-)D888 molecule to its binding site, whereas drug dissociation from the closed channel conformation is restricted by bulky channel gates. The functional data indicating a location of a trapped (-)D888 molecule close to the central pore region are supported by a homology model illustrating that the closed Cav1.2 is able to accommodate a large cation such as (-)D888.

Coupled and independent contributions of residues in IS6 and IIS6 to activation gating of CaV1.2.

Voltage dependence and kinetics of CaV1.2 activation are affected by structural changes in pore-lining S6 segments of the α1-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 (“bundle crossing region”), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S., Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol. Chem. 280, 38471–38477). Here we report that S435P in IS6 results in a large shift of the activation curve (-25.9 ± 1.2 mV) and slower current kinetics. Threonine substitutions at positions Leu-429 and Leu-434 induced a similar kinetic phenotype with shifted activation curves (L429T by -6.6 ± 1.2 and L434T by -12.1 ± 1.7 mV). Inactivation curves of all mutants were shifted to comparable extents as the activation curves. Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant cycle analysis. Double mutations in segments IS6 and IIS6 induce either additive (L429T/I781T, -34.1 ± 1.4 mV; L434T/I781T, -40.4 ± 1.3 mV; L429T/L779T, -12.6 ± 1.3 mV; and L434T/L779T, -22.4 ± 1.3 mV) or nonadditive shifts of the activation curves along the voltage axis (S435P/I781T, -33.8 ± 1.4 mV). Mutant cycle analysis revealed energetic coupling between residues Ser-435 and Ile-781, whereas other paired mutations in segments IS6 and IIS6 had independent effects on activation gating.

Molecular dynamics and mutational analysis of a channelopathy mutation in the IIS6 helix of Ca V 1.2

A channelopathy mutation in segment IIS6 of CaV1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarised voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in CaV1.2 (midpoint of activation curve (V0.5) shifted to -37.7 ± 1.2 mV). We show here that wild type gating can partially be restored by a helix stabilising rescue mutation N785A. V0.5 of I781T/N785A (V0.5 = -21.5 ± 0.6 mV) was shifted back towards wild type (V0.5 = -9.9±1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform MD-simulations on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187 - F1194) and in helix IIS6 (N785-L786) were observed. The simulated structural changes in S6 segments of I781T/N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild type CaV1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments.

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

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.