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The large-conductance, Ca2+-, and voltage-activated K+ (BK) channel consists of the pore-forming α (BKα) subunit and regulatory β and γ subunits. The γ1–3 subunits facilitate BK channel activation by shifting the voltage-dependence of channel activation toward the hyperpolarization direction by about 50–150 mV in the absence of Ca2+. We previously found that the intracellular C-terminal positively charged regions of the γ subunits play important roles in BK channel modulation. In this study, we found that the intracellular C-terminal region of BKα is indispensable in BK channel modulation by the γ1 subunit. Notably, synthetic peptide mimics of the γ1–3 subunits’ C-terminal positively charged regions caused 30–50 mV shifts in BKα channel voltage-gating toward the hyperpolarization direction. The cationic cell–penetrating HIV-1 Tat peptide exerted a similar BK channel–activating effect. The BK channel–activating effects of the synthetic peptides were reduced in the presence of Ca2+ and markedly ablated by both charge neutralization of the Ca2+-bowl site and high ionic strength, suggesting the involvement of electrostatic interactions. The efficacy of the γ subunits in BK channel modulation was reduced by charge neutralization of the Ca2+-bowl site. However, BK channel modulation by the γ1 subunit was little affected by high ionic strength and the positively charged peptide remained effective in BK channel modulation in the presence of the γ1 subunit. These findings identify positively charged peptides as BK channel modulators and reveal a role for the Ca2+-bowl site in BK channel modulation by positively charged peptides and the C-terminal positively charged regions of auxiliary γ subunits.
mechanosensitivity of TTN3 depends on the cytoskeleton integrity of the cell. Here, we found that loss of TTN3 MS current in HEK-P1KO is highly correlated with the focal adhesion (FA) complex. When the F-actin assembly is strengthened after jasplakinolide-treatment, mechanical stimuli robustly evoked MS currents in Ttn3-transfected HEK-P1KO cells. HEK-P1KO cells showed dramatic loss of FA proteins and knockdown of FA proteins in HEK cells also reduced the TTN3-dependent MS currents. Thus, we conclude that mechanosensitivity of TTN3 is dependent on the cytoskeleton integrity. This finding supports the idea that TTN3 is an essential component of the slowly inactivating MS channel complex.
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