The Effects And Mechanisms Of Ca_v1.2 Glutamate Substitution Mutant Channels On Ion Permeation And Gating

BackgroundVoltage-gated calcium channels are primary channels in myocytes and play a critical role in regulating excitation-contraction coupling and electrical signal transmitting. They also function in multiple receptors of various anti-arrhythmia medicines. Researches on the mechanisms of ion permeation and gating of voltage-gated calcium channels have been doing for a long time, and show that selectivity filter consisted with four glutamate (EEEE) has high affinity for Ca2+ binding which contribute channel ion permeation and gating mechanisms. However, since the selectivity filter couldn't explicate all the characters of channel ion permeation and gating mechanisms, many hypotheses deduced that there are other ion binding regions in voltage-gated calcium channel. But either the location or the mechanism of the possible binding regions isn't known clearly yet. We substituted the amino acids of CaV1.2 function associated region to make a series of mutant channels, then use whole-cell patch clamp to record Ca2+ and Ba2+ current to observe the effects of the mutant channels on ion permeation and gating in order to further discuss the functional mechanism of calcium channel.Part1Objective To substitute the amino acids of CaV1.2 function associated region to make a series of mutant channels to observe the affects of the mutant channels on ion permeation and gating.Methods Site-directed mutagenesis was used to sequentially replace amino acid residues in the calcicludine binding segment (Trp-1121 to Met-1136) with glutamate. Wild-type and mutant CaV 1.2 channels were expressed into cell HEK 293. Whole-cell patch-clamp recordings were used to record Ca2+ and Ba2+ currents to acquireⅠ-Ⅴrelationship cure and SSA curve. After normalized and fit, we compared Erev, slope, half-activation voltage and IBa(max)/ICa(max) of the mutant channels with wild-type to observe the effects of the mutant channels on ion permeation and gating.Results As expected, wild-type L-type calcium channels IBa(max)/ICa(max) was～2 and activation was～10 mV more positive in Ca2+ vs. Ba2+. With the exception of F1126E, the permeation and gating properties of the 13 mutant channels tested were indistinguishable from wild-type. F1126E equalized L-type calcium channel currents (IBa(max/ICa(max)≈1) and the half-activation voltage between Ca2+ and Ba2+. The half-activation voltage of Ba2+ for F1126E depolarized compared to wild-type to match that of Ca2+ which didn't change compared to wild-type.Conclusion In all the CaV1.2 functional associated amino acid substituted channels, F1126E is the only one affects ion permeation and gating of the channel. It normalizes the ion selectivity and voltage dependence differences for Ca2+ and Ba2+.Part 2Objective To study permeation mechanism for wild-type and F1126E from several approachs, as conductance, unitary current amplitude, does-response relationships of block of Li+ and saturating-conductance relationships.Methods Wild-type and mutant CaV1.2 channels were expressed into cell HEK 293. Whole-cell patch-clamp recordings were used to record Ca2+ and Ba2+ currents to acquire IIV relationship curve. After normalized and fit, we compared relative GBa, GCa and GBa/GCa of F1126E with wild-type. At the same time, we use nonstationary noise analysis to acquire the unique current amplitude of Ba2+ and Ca2+. Furthermore, we recorded the does-response relationships of Ca2+ and Ba2+ block Li+ to probe the high affinity binding of single Ba2+ and Ca2+ ions to the selectivity filter. And the relationships between saturating Ba2+ and Ca2+ conductance were measured to see the interaction of multiple divalent ions within the pore.Results As expected, the relative conductance for Ba2+ was twice as much as that of Ca2+ for wild-type, GBa/GCa≈2, however, there was no difference between GBa and GCa, GBa/GCa≈1.GBa for F1126E significantly decreaseed to match Gca which kept the same as wild-type. In addiction, unique current amplitude (i) for Ca2+ and Ba2+ acquired using nonstationary noise analysis showed that iBa reduced for F1126E to match iCa which didn't change from wild-type. Moreover, F1126E didn't alter the half-maximum of Ca2+ and Ba2+ block Li+ but it alters the relationship between saturating Ba2+ and Ca2+ conductance. The interactions of multiple Ba2+ were changed to match that of Ca2+ which stayed the same as wild-type.Conclusion F1126E diminishes ion permeation differences between Ca2+ and Ba2+ probably because the modifying of interaction of multiple Ba2+ within the pore to reduce GBa and iBa.But the mutant channel doesn't alter high affinity binding of single Ba2+ and Ca2+ ions to the selectivity filter or the permeation of Ca2+.Part 3Objective Using alanine to substitute Phe-1126 to learn the mechanism of the changing of gating made by F1126E. To record the steady-state inactivation and close-state deactivation properties to further probe the gating mechanism of Cav1.2.Methods Steady-state inactivation andⅡⅤrelationship curves of wild-type and F1126E were recorded to inquire steady-state inactivation slope, half-inactivation voltage and deactivation time constants (τ) to further discuss the gating mechanism of CaV1.2. Site-directed mutagenesis was used to replace amino acid residue Phe-1126 with alanine to make F1126A mutant channel. Wild-type and F1126A were expressed into cell HEK 293. Whole-cell patch-clamp recordings were used to record Ca2+ and Ba2+ currents to acquire the SSA curve of F1126A. And fit the curve to get half-activation voltage of Ca2+ and Ba2+ for F1126A.Results As expected, half-inactivation voltage of Ca2+ and Ba2+ has～10 mV voltage differs between Ca+ and Ba2+ for wild-type. However, no differences are shown for either half-inactivation voltage orτfor F1126E. The half-inactivation voltage of Ba2+ for F1126E further depolarizes than wild-type to match that of Ca2+ which didn't change from wild-type. At the same time, the deactivation time constants remained the same as wild-type. Half-activation voltage of F1126A demonstrates no significant difference from wild-type calcium channel.Conclusion F1126E diminishes the differences of steady-state inactivation between Ca2+ and Ba2+. Half-inactivation voltage of Ba2+ further depolarizes for F1126E to match those of Ca2+ which are the same as wild-type. Moreover, Phe-1126 does not contribute to the formation of a Ca2+ binding site in the outer vestibule of the pore.SummarySite-directed mutagenesis was used to sequentially substitute channel functional associated amino acid residues with glutamate to study the effects of the mutant channels on ion permeation and gating. Whole-cell patch-clamp recordings were used to record Ca2+ and Ba2+ currents to detect the different properties of ion permeation and gating between the two catalogues of ions in order to further discuss the mechanisms of ion permeation and gating for calcium channel. Within all 13 amino acid residues, we found only one is crucial for both ion permeation and gating. Our results prove that the two properties of ion permeation and gating are close correlated with each other of CaV1.2, more important, the outer vestibule amino acids of the channel contribute to the formation of a selective calcium binding site. The conclusions of our project are brand new basic theories for calcium channel functional mechanism studies.