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Hatouf H. Sukkarieh
Pharmacology & Physiology Program
B.Sc., 1999, Applied Science University, Jordan
Thesis Advisor: Roman Shirokov, Ph.D.
Department of Pharmacology & Physiology
Monday, July 29, 2013
10:00 A.M., MSB Room H-609
CaV1,2 channels are important players in many physiological processes. Detailed studies of molecular underpinnings of CaV1.2 function are fundamental for development of their pharmacology and therapeutics.
Current views of selectivity mechanisms in Ca2+ and Na+ voltage-gated channels focus on the structure made by loops between S5 and S6 segments of the pore-forming subunit. However, previous studies also indicated that specifics of S6 segments are important for ion permeability. Based on known crystal structures of bacterial Na+ channel, molecular dynamics modeling, and mutational studies, we propose that selectivity of Cav1.2 channels depends on residues in S6 segments. In this study, we examined the roles of three groups of highly conserved residues. One is a ring of four asparagine residues (N428, N776, N1189, and N1499I - one each in S6 segment of each domain). The other two groups comprise mostly phenylalanines in S6. According to the crystal structures they may interact with the asparagines. We produced corresponding mutants in both bacterial Na+ channel (NachBac) and CaV1.2 Ca2+ channels. Mutants were heterologously expressed in mammalian tsA201 cells for analysis of electrophysiological properties.
The asparagines were point-mutated in Cav1.2 to aspartic acid, lysine, alanine, or isoleucine. Most of the asparagine mutations did not eliminate ionic currents. However, the N428A, N428I, N776A and N1499I nearly eliminated Ca2+/Ba2+ currents. Because these mutations left gating currents unaffected, mutated channels were normally targeted to the plasma membrane. It is known that monovalent ions can pass through Ca2+channels at sub-micromolar Ca2+. The N428A and N428I did not pass monovalent ions. However, neither whole-cell, nor single-channel currents carried by monovalent ions were altered in the N776A and N1499I. In NachBac, the corresponding N225I, or N225A, eliminated Na+ current.
To investigate possible role of the asparagines in Ca2+/Na+ selectivity, we produced double mutants of the asparagines and glutamates in the selectivity locus. We studied to greater detail the E1145K/N1499I mutant that combined a 1000 fold reduction of affinity for Ca2+ in the selectivity locus with the strongest specific reduction of Ca2+ conductance. The N1499I mutation in the S6 had no significant effect on magnitudes of inward Na+ and outward Cs+ currents passing at 1 mM Ca2+ through the selectivity filter modified by the E1145K mutation.
Residues in the “upper F ring” (F424, Y772, F1185, and F1495) and the “lower F ring” (V430, F778, F1191, and F1501) were point-mutated to alanine. The V430A mutation did not produce a functional channel. Corresponding mutations in NachBac were F221A and F227A. The F to A mutations of the “upper” ring had no significant effect. However, the Y772A caused significant loss of coupling between gating and ionic currents, i.e. ionic currents were disproportionally small. Among the F to A mutants of the “lower” ring, only the F778A had significant effects, which were a -30 mV shift of activation curve and a 3-fold slowing of closing kinetics. Similar effect was observed for the corresponding F227A mutant in NachBac.
The above results show that Ca2+ selectivity/permeation of CaV1.2 channels is not structurally confined to the selectivity EEEE locus and that other residues in the permeation pathway, such as N776 and N1499 in the S6 segments of the second and fourth domains may also be involved. The findings of this work lay experimental foundation for molecular dynamics model of CaV1.2 that is being developed in the lab. According to it, the asparagines face away from the center of the channel. Unexpectedly, N428, N776 and N1499, but not N1189, are in water-filled crevices on the intracellular side of the channel. How this is important for specific Ca2+, but not Na+, permeation is under investigation. The model also proposes that the “lower” F778 in the second domain of Cav1.2 obstructs the permeation pathway and, thus, destabilizes the open conformation. The corresponding F227 in NachBac might act similarly.