The large-conductance Ca(2+)-activated K(+) channel interacts with the apolipoprotein ApoA1

Abstract

Owing to the multifaceted functions of the large conductance Ca(2+)-activated K(+) channel (BK), identification of protein-protein interactions is essential in determining BK regulation. A yeast two-hybrid screening of a cochlear cDNA library revealed a BK-ApoA1 interaction. Patch clamp recordings of excised membrane patches from transfected HEK293 cells showed that ApoA1 inhibits the BK alpha-subunit by significantly increasing activation and deactivation times, and shifting half-activation voltage to more positive potentials. Reciprocal coimmunoprecipitations verified the BK-ApoA1 interaction using excised sensory epithelium and ganglia. Additionally, immunocolocalization studies revealed BK and ApoA1 expression in both receptor cells and auditory neurons. These data suggest new avenues of investigation, given the importance of apolipoproteins in neurological diseases.

Fig. 1

ApoA1 increases BK half-activation voltage. (A) Currents recorded from isolated inside-out patches of HEK293 cells transfected with BK only or BK+ApoA1. Patches were stepped from −20 mV to 130 mV from a holding potential of −50 mV. Tail currents were generated by a final step back to −50 mV. (B) Average, normalized G-V curves were generated for patches containing BK-only (n = 8) and BK+ApoA1 (n = 9). Individual G-V curves were fit to Boltzmann equations and fit parameters averaged. (C) Plots of the average G_max, half-activation voltage (V_1/2), and Boltzman slope factor (Vc) are shown for the two-channel configurations. (*p < 0.05).

Fig. 2

ApoA1 slows BK kinetics. (A) Exemplar of activation currents for a voltage step from −50 mV to 100 mV. Traces were normalized to steady-state maxima after correcting for linear leak and capacitative transients. Single exponential fits (grey lines) are 6.03 and 10.32 ms for BK only and BK+ApoA1, respectively. (B) Average activation time constants plotted against the activation voltage command and fit with a single exponential show an ∼3 ms shift in BK activation with ApoA1 present. (C) Exemplar deactivation currents for a voltage step from 130 mV to −50 mV. Traces were normalized and fit as in A. Single exponential fits (grey lines) are 1.29 and 2.85 ms for BK only and BK+ApoA1, respectively. (D) Deactivation kinetics at a single voltage level of −50 mV were averaged from single exponential fits to tail currents elicited from a 130 mV activation step. Deactivation increased ∼2-fold with ApoA1. Error bars indicate one SEM. (*p < 0.05)

Fig. 3

Verification of BK/ApoA1 interactions using reciprocal coIP. (A) CoIP of BKα with ApoA1 (lane 1) and IP of BKα (lane 2) reveal prominent bands at ∼120 kDa, the expected weight of chick BKα. (B) Reciprocal coIP of ApoA1 with BKα (lane 1) and IP of ApoA1 alone (lane 2) reveal a prominent band at ∼28 kDa, the expected weight of ApoA1. The control for both panels consisted of mixing IgG beads and lysate in the absence of an antibody (lane 3). The 55 kDa band is heavy IgG, and occurs when either monoclonal or polyclonal antibodies are used to both precipitate proteins and probe the blots. “M” denotes the standard.

Fig. 4

ApoA1 (green) and BKα (red) are colocalized in chick cochlea. The colocalization of ApoA1 and BK is visualized as punctate yellow fluorescence in (A) basolateral aspects of hair cells and (B) VIIIth nerve ganglion cell body. Images are from one section of a Z-stack acquired using a step size of 0.2 μm.