We have previously reported potent activation of large-conductance Ca2+-activated K+ channels (Maxi-K) in thoracic rat aorta smooth muscle (SM) by plasmonic gold nanocrystals (AuNCs) ~of 5 nm core size having plasmon resonance of 532 nm. Green laser irradiation facilitated this effect and caused SM relaxation (1, 2). The aim of this study was to uncover the intrinsic mechanisms of Maxi-K regulation by AuNCs. Maxi-K currents in isolated rat aortic and pulmonary artery myocytes were recorded at room temperature in the whole-cell and cell-attached configurations. Maxi-K channels are both Ca2+ and voltage-sensitive, hence we compared the effects of NCs on Maxi-K channel activity under weak (0.3 mM EGTA) or strong (10 mM BAPTA/4.6 mM Ca2+) to “clamp” the intracellular Ca2+ concentration at 100 nM). With weak Ca2+ buffering, Maxi-K current density at 70 mV was increased by AuNCs applied at 10-4 M from 50±5 pA/pF to 130±6 pA (P<0.001; n=6), and further increased to 205.3±7.6 pA (n=6; P<0.001) by laser illumination. The potentiating effect of AuNCs alone was due to an increase in maximal conductance (Gmax) by about 50% without any shift of the activation curve of K+ conductance (the potential of half-maximal activation V1/2 was -89.6±2.4 mV and -91.02±1.99 mV in control and in the presence of AuNCs, respectively, n=6). Interestingly, green laser illumination (5 mW, 532 nm) had no significant effect on the maximal K+ conductance (2.09±0.08 and 2.18±0.06 nS/pF before and after laser illumination, n=6), but instead shifted the V1/2 value to -79.6±1.5 mV, n=6; P<0.05). Under conditions of strong intracellular Ca2+ buffering, no effect of AuNCs on current density was observed (54±4 pA/pF in control vs 55±4 in the presence of 10-4M AuNCs; p>0.05, n=6). Gmax and the V1/2 values also remained largely unchanged. At the single channel level, the effects of AuNCs were due to a significant increase in channel open probability, while single channel conductance remained unchanged. Intriguingly, the independent gating of maxi-K channels (e.g. binomial distribution test) was not observed, suggesting that plasmon resonance may not equally affect all channels in the patch, which is different from other common types of drug action.We conclude that AuNCs activate Maxi-KCa channels via both Gmax increase and a negative V1/2 shift. These processes are clearly calcium-dependent, as the potentiating effect of AuNCs could be completely abolished by “clamping” the intracellular Ca2+ concentration at 100 nM. This is an important step towards not only better understanding of the nature of this effect, but also the development of new methods of nano-photonic control of voltage-gated ion channels function in living cells that, in turn, may have significant promise for developing next generation of ion channel modulators with high target specificity and low toxicity.