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Permeant ion binding affinity in subconductance states of an L-type Ca²⁺ channel expressed in Xenopus laevis oocytes

Robin K. Cloues and William A. Sather

1. The relationship between single-channel conductance and ion binding affinity in Ca²⁺ channels was investigated by measuring differences in the apparent binding affinity (K'_D) for Ca²⁺ among naturally occurring conductance states of an L-type (_1C) Ca²⁺ channel heterologously expressed in Xenopus oocytes. Using cell-attached patch recordings, three or more conductance levels were observed when Ca²⁺, Ba²⁺ or Li⁺ was used as the permeating ion.

2. With Li⁺ as the charge carrier, low concentrations of Ca²⁺ (0·1-3·0 µM) produced discrete blocking events in all conductance states. Measurements of open and blocked times as a function of Ca²⁺ concentration were used to calculate rates of block and unblock.

3. K'_D was calculated for three of the conductance levels. Binding affinity for Ca²⁺ increased as conductance decreased (K'_D: large = 7·5 µM, medium = 4·0 µM, small = 2·7 µM). The lower K'_D values of the smaller conductance states arose from a combination of larger on-rates and smaller off-rates.

4. These results imply that permeant ions such as Ca²⁺ have both easier access to, and longer dwell time in, the Ca²⁺ binding locus in the pore when the channel opens to a subconductance level as compared to the fully open level.

5. The difference in K'_D between the large and small conductance levels corresponds to a small difference in the free energy of binding, G 1k_BT, where k_B is Boltzmann's constant and T is absolute temperature (kelvin). Nonetheless, an Eyring model of Ca²⁺ channel permeation incorporating the state-specific on- and off-rate constants for Ca²⁺ was able to reproduce the large difference in channel conductance, indicating that small differences in binding energy may be able to account for large differences in amplitude between conductance states.

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