ABSTRACTHeat tolerance of heart rate in fish is suggested to be limited by impaired electrical excitation of the ventricle due to the antagonistic effects of high temperature on Na+(INa) and K+(IK1) ion currents (INais depressed at high temperatures while IK1is resistant to them). To examine the role of Na+channel proteins and the lipid matrix of the channels in heat tolerance of INa, we compared temperature-dependencies of zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) ventricular INa, and INagenerated by the cloned zebrafish and rainbow trout NaV1.4 and NaV1.5 Na+channels in HEK cells. Whole-cell patch clamp recordings showed that zebrafish ventricular INahas better heat tolerance and slower inactivation kinetics than rainbow trout ventricular INa. In contrast, heat tolerance and inactivation kinetics of zebrafish and rainbow trout NaV1.4 channels are similar when expressed in the identical plasma membrane lipid matrix of HEK cells. The same applies to NaV1.5 channels. Thermal adaptation of the ventricular INais largely achieved by differential expression of Na+channel alpha subunits: zebrafish which tolerate well high temperatures mainly express the slower NaV1.5 isoform, while rainbow trout which prefer cold waters mainly express the faster NaV1.4 isoform. Differences in elasticity (stiffness) of the lipid bilayer may be also involved in thermal adaptation of INa. These findings suggest that both the protein component and its lipid bilayer matrix are involved in thermal adaptation of the voltage-gated Na+channels and therefore in heart rate regulation under thermal stress in fish.