It is well known that voltage-gated calcium channels select for Ca²⁺ ions over other cations via binding at a high affinity binding site, formed in large part by four negatively charged residues (EEEE or EEDD, selectivity filter).  While this mechanism makes for channels highly selective for divalent cations, it has also been shown that these channels readily pass monovalent cations in the absence of divalents.  In fact under physiological conditions, where [Ca²⁺]_i is low, the current voltage relationship is highly nonlinear because inward currents are carried by Ca²⁺ and outward currents are carried by monovalent cations.  Almost all divalent cations block permeation through Ca²⁺ channels.  However, it is unclear whether this block occurs at the region of the selectivity filter and/or at other points along the permeation pathway.  In order to better understand permeation through T-type calcium channels, we used Mg²⁺ as a probe. Under conditions of symmetrical Li⁺, a permeant monovalent cation, both [Mg²⁺]_i and [Mg²⁺]_o voltage dependently reduced Li⁺ currents.  Interestingly, extreme potentials allowed “punch-through” of Mg²⁺ ions, i.e. the energy of the field helped Mg²⁺ permeate.  Consistent with the hypothesis that Mg²⁺ is able to permeate T-channels, we were able to detect inward Mg²⁺ currents in the presence of 20mM [Mg²⁺]_o.  Using a model that allowed for both block and permeation, [Mg²⁺]_o has 100-fold higher affinity than [Mg²⁺]_i.  The difference in affinity reveals asymmetry along the permeation pathway and future experiments will determine if this is due to multiple binding sites or a difference in the accessibility to a single binding site. 

09/04/07