FIGURE 5.

Abolition of STREX palmitoylation by 2-BP augments PKC-dependent inhibition in cells co-expressing STREX and ZERO BK channels. A, PCR products without (ZERO) and with the 174 bp STREX exon are detected in GH3/B6 cells. B and C, whole-cell outward currents (I_out) were elicited from a holding potential of −10 mV by depolarizing GH3/B6 cells every 5 s for 300 ms from −60 to +80 mV. Bars represent means ± S.E. of current densities at +80 mV. It is shown that the inhibitory effect of the PKC activator PMA (100 nm) is markedly enhanced in 2-BP-treated cells, whereas the inactive phorbol ester analog 4α-PMA is ineffective. Note that more than 90% of I_out is blocked by the specific BK channel blocking peptide iberiotoxin (IbTX, 300 nm). Numbers within bars indicate number of cells. The original current recordings are at +80 mV with interventions as indicated. The pipette solution contained 0.3 μm Ca²⁺. D and E, conductance-voltage relationships obtained from inside-out membrane patches of HEK293 cells co-transfected with equal amounts of plasmid DNA encoding for the ZERO and the STREX variant of the BK channel, respectively. The PKC-dependent inhibition (20 nm PKC_c, D) of G_m was almost doubled in cells treated with 2-BP (E). Means ± S.E. when exceeding the size of the symbol are shown. Insets, representative macroscopic currents at +80 mV reflecting interventions as indicated by the symbols in the corresponding figure. Numbers within bars represent the number of membrane patches. The intracellular (bath) Ca²⁺ concentration was 1 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control (Ctr) in (B–E).