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.
Cloues
R K
RK
Department of Pharmacology and Neuroscience Center, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Sather
W A
WA
D000818:Animals; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D004553:Electric Conductivity; D005260:Female; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins / Q000378:metabolism; D019789:Tetraethylammonium / Q000494:pharmacology; D014982:Xenopus laevis
D020746:Calcium Channels, L-Type; D011994:Recombinant Proteins; D019789:Tetraethylammonium