The drug efflux pump P-glycoprotein possesses two common and often linked polymorphisms that result in variable drug action. G2677T results in A893S, whereas C3435T is synonymous and has been reported to alter protein folding. We tested the effect of these MDR1 variants on Human Ether-Related A Go-Go (HERG) block by ibutilide in CHO cells 48 h following transient transfection with an IRES-dsRed vector containing MDR1, G2677T MDR1, G2677T/C3435T MDR1 or an empty bicistronic site and an IRES-GFP vector containing HERG (KCNH2). Cotransfection of MDR1 variants had no effect on I(Kr) amplitude at baseline. Cells cotransfected with MDR1-G2677T showed resistance to ibutilide vs HERG alone (IC(50): 105.3+/-1.42 nM vs 27.4+/-2.5 nM; P<0.0001), consistent with the idea that A893S attenuates I(Kr) block by enhancing drug efflux and thus reducing the drug available to interact with the channel binding site. However, G2677T/C3435T cells showed ibutilide sensitivity similar to cells expressing HERG alone (IC(50): 22.2+/-0.9 nM). Immunostaining showed that the C3435T variant did not traffic to the cell surface. Coculture with fexofenadine(1 microM), an MDR1 substrate known to rescue misfolding in other membrane proteins, restored cell surface expression of MDR1 G2677T/C3435T and restored resistance to block HERG by ibutilide 200 nM (98.5+/-0.98% vs 42.3+/-2.2%, P<0.001). The non-synonymous MDR1 variant G2677 T (A893S) confers resistance to ibutilide block of I(Kr), which is mitigated by the C3435T polymorphism through reduced protein expression, an effect that can be restored by coculture with fexofenadine. These data identify ibutilide as an MDR1 substrate and further support the concept that variable drug transport function can modulate the action of HERG blockers.