The mechanisms of verapamil and tetraethylammonium (TEA) inhibition of voltage-gated K+ channels in LNCaP human prostate cancer cells were studied in whole-cell and outside/insideout patch-clamp configurations. Rapidly activating outward K+ currents (IK) exhibited neither C-type, nor rapid (human ether á go-go-related gene-type)inactivation. With 2 mM [Mg2+]o, IK activation kinetics was independent of holding potential, suggesting the absence of ether á go-go-type K+ channels. Extracellular applications of TEA and verapamil (IC50 = 11 μM) rapidly (12 s) inhibited IK in LNCaP cells. Blocking was also rapidly reversible. Intracellular TEA (1 mM), verapamil (1 mM), and membrane-impermeable N-methyl-verapamil (25 μM) did not influence whole-cell IK, although both phenylalkylamines inhibited single-channel currents in inside-out patches. Extracellular application of N-methyl-verapamil (25 μM) had no influence on IK. Our results are compatible with the hypothesis that, in LNCaP cells expressing C-type inactivation-deficient voltage-activated K+ channels, phenylalkylamines interact with an intracellular binding site, and probably an additional hydrophobic binding site that does not bind charged phenylalkylamines. The inhibiting effects of verapamil and TEA on IK were additive, suggesting independent K+-channel blocking mechanisms. Indeed, TEA (1 mM) reduced a single-channel conductance (from 7.3 ± 0.5 to 3.2 ± 0.4 pA at a membrane potential of +50 mV, n = 6), whereas verapamil (10 μM) reduced an open-channel probability (from 0.45 ± 0.1 in control to 0.1 ± 0.09 in verapamil-treated cells, n = 9). The inhibiting effects of verapamil and TEA on LNCaP cell proliferation were not multiplicative, suggesting that both share a common antiproliferative mechanism initiated through a K+ channel block.
ASJC Scopus subject areas
- Molecular Medicine