Phospholamban (PLN) and sarcolipin (SLN) are two single pass membrane proteins that regulate the sarcoplasmic reticulum Ca 2+-ATPase (SERCA). Given their high propensity for oligomerization, both proteins have been hypothesized to form selective ion channels in the sarcoplasmic reticulum membrane. Here, this hypothesis was tested by using a novel and general method for determining the minimum transmembrane potential required for a peptide or a membrane protein to form a cation-conducting pore across a lipid bilayer. The method uses a mercury-supported biomimetic membrane, consisting of a lipid bilayer incorporating the protein and tethered to the mercury surface via a disulfidated tetraethyleneoxy hydrophilic spacer. The biomimetic membrane, immersed in aqueous KCl, is subjected to a series of progressively more negative potential jumps, starting from a fixed, slightly positive transmembrane potential. If the protein can form a K +-conducting pore, a final potential is ultimately attained at which the protein triggers a flux of K + ions that saturates the hydrophilic spacer, as revealed by a well-defined sigmoidal charge step of 45 to 60 μC cm -2 in the charge vs. time curve following the negative potential jump. Potential jumps negative enough to attain non-physiological final transmembrane potentials induce SLN and its T18A and T5A mutants, as well as pentameric PLN, its phosphorylated form (pPLN), and its AFA-PLN mutant, to saturate the hydrophilic spacer with K + cations. We conclude that PLN, pPLN and AFA-PLN, which contain a completely hydrophobic sequence in the transmembrane domain, are able to transport ions only when subjected to non-physiological transmembrane potentials.