Repolarization and termination of the ventricular cardiac action potential is highly dependent on the activation of the slow delayed-rectifier potassium I Ks channel. Disruption of the I Ks current leads to the most common form of congenital long QT syndrome (LQTS), a disease that predisposes patients to ventricular arrhythmias and sudden cardiac death. We previously demonstrated that polyunsaturated fatty acid (PUFA) analogues increase outward K + current in wild type and LQTS-causing mutant I Ks channels. Our group has also demonstrated the necessity of a negatively charged PUFA head group for potent activation of the I Ks channel through electrostatic interactions with the voltage-sensing and pore domains. Here, we test whether the efficacy of the PUFAs can be tuned by the presence of different functional groups in the PUFA head, thereby altering the electrostatic interactions of the PUFA head group with the voltage sensor or the pore. We show that PUFA analogues with taurine and cysteic head groups produced the most potent activation of I Ks channels, largely by shifting the voltage dependence of activation. In comparison, the effect on voltage dependence of PUFA analogues with glycine and aspartate head groups was half that of the taurine and cysteic head groups, whereas the effect on maximal conductance was similar. Increasing the number of potentially negatively charged moieties did not enhance the effects of the PUFA on the I Ks channel. Our results show that one can tune the efficacy of PUFAs on I Ks channels by altering the pK a of the PUFA head group. Different PUFAs with different efficacy on I Ks channels could be developed into more personalized treatments for LQTS patients with a varying degree of I Ks channel dysfunction.