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

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 (alpha1C) 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 microM) 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 microM, medium = 4.0 microM, small = 2.7 microM). 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, DeltaDeltaG approximately 1kBT, where kB 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.