G628S is a mutation in the signature sequence that forms the selectivity filter of the human ether-a-go-go-related gene (hERG) channel (GFG) and is associated with long-QT2 syndrome. G628S channels are known to have a dominant-negative effect on hERG currents, and the mutant is therefore thought to be nonfunctional. This study aims to assess the physiological mechanism that prevents the surface-expressing G628S channels from conducting ions. We used voltage-clamp fluorimetry along with two-microelectrode voltage clamping in Xenopus oocytes to confirm that the channels express well at the surface, and to show that they are actually functional, with activation kinetics comparable to that of wild-type, and that the mutation leads to a reduced selectivity to potassium. Although ionic currents are not detected in physiological solutions, removing extracellular K(+) results in the appearance of an inward Na(+)-dependent current. Using whole-cell patch clamp in mammalian transfected cells, we demonstrate that the G628S channels conduct Na(+), but that this can be blocked by both intracellular and higher-than-physiological extracellular K(+). Using solutions devoid of K(+) allows the appearance of nA-sized Na(+) currents with activation and inactivation gating analogous to wild-type channels. The G628S channels are functionally conducting but are normally blocked by intracellular K(+).