We have isolated an hSK3 isoform from a human embryonic cDNA library that we have named hSK3_ex4. This isoform contains a 15 amino acid insertion within the S5 to P-loop segment. Transcripts encoding hSK3_ex4 are coexpressed at lower levels with hSK3 in neuronal as well as in non-neuronal tissues. To investigate the pharmacokinetic properties of hSK3_ex4, we expressed the isoforms hSK3 and hSK3_ex4 in tsA cells. Both isoforms were similarly activated by cytosolic Ca2+ (hSK3, EC50=0.91 +/- 0.4 microM; hSK3_ex4, EC50=0.78 +/- 0.2 microM) and by 1-ethyl-2-benzimidazolinone (hSK3, EC50=0.17 mM; hSK3_ex4, 0.19 mM). They were both blocked by tetraethylammonium (hSK3, Kd=2.2 mM; hSK3_ex4, 2.6 mM) and showed similar permeabilities relative to K+ for Cs+ (hSK3, 0.17 +/- 0.04, n=3; hSK3_ex4, 0.17 +/- 0.05, n=3) and Rb+ (hSK3, 0.79 +/- 0.04, n=3; hSK3_ex4, 0.8 +/- 0.07, n=3). Ba2+ blocked both isoforms, and in both cases, the block was strongest at hyperpolarizing membrane potentials. However, the voltage-dependence of hSK3 was stronger than that of hSK3_ex4. The most obvious distinguishing feature of this new isoform was that whereas hSK3 was blocked by apamin (Kd=0.8 nM), scyllatoxin (Kd=2.1 nM), and d-tubocurarine (Kd=33.4 microM), hSK3_ex4 was not affected by apamin up to 100 nM, scyllatoxin up to 500 nM, and d-tubocurarine up to 500 microM. So far, isoform hSK3_ex4 forms the only small-conductance calcium-activated potassium (SK) channels, which are insensitive to the classic SK blockers.