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 affi nity in Ca2+ channels was investigated by measuring differences in the appa rent binding affinity (K'(D)) for Ca2+ among naturally occurring conductanc e states of an L-type (alpha(1C)) Ca2+ channel heterologously expressed in Xenopus oocytes. Using cell-attached patch recordings, three or more conduc tance levels were observed when Ca2+, Ba2+ or Li+ was used as the permeatin g ion. 2. With Li+ as the charge carrier, low concentrations of Ca2+ (0.1-3.0 mu M ) produced discrete blocking events in all conductance states. Measurements of open and Mocked 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 affini ty for Ca2+ increased as conductance decreased (K'(D): large = 7.5 mu M, me dium = 4.0 mu M, small = 2.7 mu M). The lower K'(D) values of the smaller c onductance states arose from a combination of larger on-rates and smaller o ff-rates. 4. These results imply that permeant ions such as Ca2+ have both easier acc ess to, and longer dwell time in, the Ca2+ binding locus in the pore when t he channel opens to a subconductance level as compared to the fully open le vel. 5. The difference in K'(D) between the large and small conductance levels c orresponds to a small difference in the free energy of binding, Delta Delta G approximate to 1k(B)T, where k(B) is Boltzmann's constant and T is absol ute temperature (kelvin). Nonetheless, an Eyring model of Ca2+ channel perm eation incorporating the state-specific on- and off-rate constants for Ca2 was able to reproduce the large difference in channel conductance, indicat ing that small differences in binding energy may be able to account for lar ge differences in amplitude between conductance states.