Single Channel Analysis of the Regulation of GIRK1/GIRK4 Channels by Protein Phosphorylation

G-Protein activated, inwardly rectifying potassium channels (GIRKs) are important effectors of G-protein β/γ-subunits, playing essential roles in the humoral regulation of cardiac activity and also in higher brain functions. G-protein activation of channels of the GIRK1/GIRK4 heterooligomeric composition is controlled via phosphorylation by cyclic AMP dependent protein kinase (PKA) and dephosphorylation by protein phosphatase 2A (PP(2)A). To study the molecular mechanism of this unprecedented example of G-protein effector regulation, single channel recordings were performed on isolated patches of plasma membranes of Xenopus laevis oocytes. Our study shows that: (i) The open probability (P(o)) of GIRK1/GIRK4 channels, stimulated by coexpressed m(2)-receptors, was significantly increased upon addition of the catalytic subunit of PKA to the cytosolic face of an isolated membrane patch. (ii) At moderate concentrations of recombinant G(β1/γ2), used to activate the channel, P(o) was significantly reduced in patches treated with PP(2)A, when compared to patches with PKA-cs. (iii) Several single channel gating parameters, including modal gating behavior, were significantly different between phosphorylated and dephosphorylated channels, indicating different gating behavior between the two forms of the protein. Most of these changes were, however, not responsible for the marked difference in P(o) at moderate G-protein concentrations. (iv) An increase of the frequency of openings (f(o)) and a reduction of dwell time duration of the channel in the long-lasting C(5) state was responsible for facilitation of GIRK1/GIRK4 channels by protein phosphorylation. Dephosphorylation by PP(2)A led to an increase of G(β1/γ2) concentration required for full activation of the channel and hence to a reduction of the apparent affinity of GIRK1/GIRK4 for G(β1/γ2). (v) Although possibly not directly the target of protein phosphorylation/dephosphorylation, the last 20 C-terminal amino acids of the GIRK1 subunit are required for the reduction of apparent affinity for the G-protein by PP(2)A, indicating that they constitute an essential part of the off-switch

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

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 (α1C) 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.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.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.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.The difference in K'D between the large and small conductance levels corresponds to a small difference in the free energy of binding, ΔΔG≈ 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