Strukturdeterminanten der Aktivierung von L-Typ Kalziumkanälen in den Segmenten IS6 und IIS6

Als Ziel der vorliegenden Arbeit wurde der Einfluss von Mutationen in porenbildenden S6 Segmenten auf das „gating“ Verhalten von CaV1.2 untersucht. Besonders signifikante Effekte wurden durch Prolin- bzw. Threoninsubstitutionen im unteren Drittel („bundle crossing region“) der Segmente IS6 und IIS6 induziert. Hydrophobe Interaktionen zwischen S6 Segmenten oder Nachbarregionen sind an der Stabilisierung der geschlossenen Kanalkonformation beteiligt (Hohaus et al., 2005; Kudrnac et al., 2009). Diese Hypothese wurde durch Mutationen mit Aminosäuren unterschiedlicher Hydrophobizität, Größe und Polarität erhärtet. Die Verschiebung der spannungsabhängigen Aktivierung ging mit einer verlangsamten Aktivierung bei hyperpolarisierten Potentialen, einer verlangsamten Dekativierung und verminderter Inaktivierung einher. Subsitutionen von Ile781 können sowohl die geschlossene Konformation destabilisieren als auch die offene Konformation von CaV1.2 stabilisieren. Außerdem könnte an den Positionen Cys779–Ala782 eine flexible Position für „helix bending“ vorliegen. Auch Prolinsubstitution S435P in IS6 führte zu einer starken Verschiebung der Aktivierungskurve und verlangsamter Stromkinetik. Die Threoninsubstitutionen Leu429 und Leu434 führten zu einem ähnlichen kinetischen Phänotyp mit verschobenen Aktivierungskurven. Die entsprechenden Inaktivierungskurven aller Mutanten wurden proportional in negativer Richtung auf der Potentialachse verschoben. Eine Abhängigkeit von strukturellen Veränderungen in IS6 und IIS6 wurde mittels „mutant cylce analysis“ analysiert. Doppelmutationen in diesen S6 Segmenten induzieren entweder additive oder nicht-additive Verschiebungen der Aktivierungskurven entlang der Potentialachse. Die „mutant cycle analysis“ ergab eine energetische Kopplung zwischen den Aminosäuren S435 und I781; weitere Doppelmutationen in IS6 und IIS6 zeigten unabhängige Effekte auf das Aktivierungsverhalten von CaV1.2

Different pathways for activation and deactivation in CaV1.2: a minimal gating model

Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants α and β, and γ and δ, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 CaV1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the “pore-releasing” sensor movement ((x(V)) was much weaker than the voltage dependence of “pore-locking” sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening CaV1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain

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

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