The large conductance Ca(2+) -activated K(+) (BKCa) channel regulates cell proliferation in SH-SY5Y neuroblastoma cells by activating the staurosporine-sensitive protein kinases.

Abstract

Here we investigated on the role of the calcium activated K+-channels(BKCa) on the regulation of the neuronal viability. Recordings of the K+-channel current were performed using patch-clamp technique in human neuroblastoma cells (SH-SY5Y) in parallel with measurements of the cell viability in the absence or presence of the BKCa channel blockers iberiotoxin(IbTX) and tetraethylammonium (TEA) and the BKCa channel opener NS1619. Protein kinase C/A (PKC, PKA) activities in the cell lysate were investigated in the presence/absence of drugs. The whole-cell K+-current showed a slope conductance calculated at negative membrane potentials of 126.3 pS and 1.717 nS(n = 46) following depolarization. The intercept of the I/V curve was −33 mV. IbTX(10−8 – 4 × 10−7 M) reduced the K+-current at +30 mV with an IC50 of 1.85 × 10−7 M and an Imax of −46% (slope = 2.198) (n = 21). NS1619(10–100 × 10−6 M) enhanced the K+-current of +141% (n = 6), at −10 mV(Vm). TEA(10−5–10−3 M) reduced the K+-current with an IC50 of 3.54 × 10−5 M and an Imax of −90% (slope = 0.95) (n = 5). A concentration-dependent increase of cell proliferation was observed with TEA showing a maximal proliferative effect(MPE) of +38% (10−4 M). IbTX showed an MPE of +42% at 10−8 M concentration, reducing it at higher concentrations. The MPE of the NS1619(100 × 10−6 M) was +42%. The PKC inhibitor staurosporine (0.2–2 × 10−6 M) antagonized the proliferative actions of IbTX and TEA. IbTX (10 × 10−9 M), TEA (100 × 10−6 M), and the NS1619 significantly enhanced the PKC and PKA activities in the cell lysate with respect to the controls. These results suggest that BKCa channel regulates proliferation of the SH-SY5Y cells through PKC and PKA protein kinases.