Abstract
The auxiliary beta4 subunit of the human slowpoke calciumdependent potassium (slo) channel is expressed predominantly in the brain. Co-expression of beta4 subunit with the slo channel alpha subunit in HEK293 and Chinese hamster ovary cells slows channel activation and deactivation and also shifts the voltage dependence of the channel to more depolarized potentials. We show here that the functional interaction between the hbeta4 subunit and the slo channel is influenced by the phosphorylation state of hbeta4. Treatment of cells with okadaic acid (OA) reduces the effect of hbeta4 on slo channel activation kinetics and voltage dependence but not on slo channel deactivation kinetics. The effect of OA can be blocked by mutating three putative serine/threonine phosphorylation sites in hbeta4 (Thr-11/Ser-17/Ser-210) to alanines, suggesting that OA potentiates phosphorylation of hbeta4 and thereby suppresses its functional coupling to the slo channel. Mutation of Ser-17 alone to a negatively charged residue (S17E) can mimic the effect of OA. Mutating all three phosphorylation sites in hbeta4 to negatively charged residues (T11D/S17E/S210E) not only suppresses the effect of hbeta4 on slo channel activation kinetics and voltage dependence, it also suppresses its effect on slo channel deactivation kinetics. Co-immunoprecipitation/Western blot experiments indicate that all of these hbeta4 mutants, as well as the wild-type hbeta4, bind to the slo channel. Taken together, these data suggest that phosphorylation of the beta4 subunit dynamically regulates the functional coupling between the beta4 subunit and the pore-forming alpha subunit of the slo channel. In addition, phosphorylation of different residues in hbeta4 differentially influences its effects on slo channel activation kinetics, deactivation kinetics, and voltage dependence.
Highlights
Large conductance Ca2ϩ-dependent potassium (KCa or maxi K) channels are ubiquitously expressed in neurons and many other tissues
Mutating all three phosphorylation sites in h4 to negatively charged residues (T11D/Ser-17 alone to a negatively charged residue (S17E)/S210E) suppresses the effect of h4 on slo channel activation kinetics and voltage dependence, it suppresses its effect on slo channel deactivation kinetics
Similar modulation of slo channel activation by 4 was observed in the whole-cell recording configuration (Fig. 1, A and B). This effect of 4 on slo channel activation kinetics gradually decreased with time after going into whole-cell mode
Summary
Cloning and Transient Expression in CHO1 Cells—h4 was cloned into the pIRES2-EGFP vector (CLONTECH), a bicistronic vector that allows co-expression of h4 and green fluorescent protein in the same cell. Cells were transfected with both constructs, and transfected cells were identified by their green fluorescence; all such cells were found to express slo current. For measuring the time course of slo channel activation and deactivation, the cells were held at Ϫ80 mV, and the membrane potential was stepped to ϩ40 mV for 200 ms and back to Ϫ80 mV. For measuring the current-voltage relationship of the slo channel, the cells were held at Ϫ80 mV, and depolarizations in steps of 20 mV were applied for 200 ms. Horseradish peroxidase-coupled donkey anti-rabbit IgG (Amersham Biosciences) was used as the secondary antibody for hSlo blots. 32P Labeling of 4 —HEK293 cells were transfected with hSlo and h4 as described earlier. The cells were labeled for 12 h with 32P and lysed. hSlo was immunoprecipitated, and the immunoprecipitated proteins were separated on a polyacrylamide gel. 32P-Labeled proteins, including h4, were detected by autoradiography
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