I studied two aspects of permeation of the voltage-gated calcium (Ca) channel CaV 3.1, a T-type Ca channel, i.e. the concentration dependence of conductance for Ca and barium (Ba), and the voltage dependence of block by magnesium (Mg), and I also created a kinetic gating model for the channel. (1) Macroscopic CaV3.1 currents have been reported to be larger in Ca than in Ba, however, T-channels have been shown to have a similar single channel conductance for Ca and Ba in high divalent concentrations. I found that a difference in KM, but similar maximal single channel conductance for Ca and Ba, produced a larger macroscopic conductance in Ca compared to Ba at low divalent concentrations and similar macroscopic conductance at high divalent concentrations. The macroscopic currents under all conditions were larger in Ca compared to Ba because of a difference in the apparent reversal potential. (2) L-type Ca channels have asymmetric affinities for Mg when applied intracellularly compared to extracellularly. I examined the relative affinity for both extracellular and intracellular Mg for CaV3.1 in the presence of symmetrical lithium to determine whether T-channels also display asymmetry in their affinity for Mg. I found that both extracellular and intracellular Mg voltage dependently blocked the channel. However, the concentrations required to produce a similar level of block were approximately 200-fold lower for extracellular Mg compared with intracellular Mg. At extreme potentials there was a voltage dependent relief of block suggesting that the blocker can exit to the opposite side of the channel, although it was not possible to record Mg current directly. A single site model was insufficient to describe the data because of the large difference in affinity from either side. A two-site three-barrier Eyring model in which the transition states are not halfway between the wells could describe experimental data from four concentrations of Mg (two inside, two outside). (3) I also developed a kinetic gating model for CaV 3.1. The model has 5 closed states, an open state, and three inactivated states. The parameters for the model were optimized using a set of macroscopic current data and on-gating currents over a range of potentials. The model recapitulated the features of the macroscopic current data and gating current data. Using parameters in which the O→C transition was either voltage dependent or independent produced very different predictions for the single channel events and tail current timecourse. These data suggest that incorporation of single channel data and tail current timecourse will provide essential information for future model development. The basic level of understanding developed here of how these Ca channels work is a crucial starting point to understanding the diverse cellular processes in which these channels are involved.
|Commitee:||Fox, Aaron, Grosman, Claudio, Nelson, Deborah, Perozo, Eduardo|
|School:||The University of Chicago|
|School Location:||United States -- Illinois|
|Source:||DAI-B 70/08, Dissertation Abstracts International|
|Keywords:||Barium, Block, Calcium channels, Conductance, Magnesium, Single-channel|
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