Channel cytoplasmic loops alter voltage-dependent sodium channel activation in an isoform-specific manner.

1. The isoform-specific functional role of cytoplasmic structures of two voltage-gated sodium channel isoforms, the human cardiac channel (hH1) and the adult human skeletal muscle channel (hSkM1) was investigated through functional comparison of chimeras. 2. The voltage of half-activation (V(a)) for hH1 was shifted by > 20 mV in the hyperpolarised direction following internal papain treatment ('papain sensitive'), while V(a) for hSkM1 was unaffected ('papain insensitive'). 3. The hH1 region(s) responsible for this papain sensitivity was localised by testing a series of hH1/hSkM1 chimeras in which combinations of the large hH1 cytoplasmic loops joining the four transmembrane domains replaced analogous hSkM1 loops. Various chimeras were used to determine the smallest subset of loops that converted fully the papain-insensitive hSkM1 into a papain-sensitive channel. Then three converse chimeras were tested in which hSkM1 loops replaced hH1 loops to determine the smallest subset of loops necessary and sufficient to convert the papain-sensitive hH1 into a papain-insensitive channel. 4. Functional studies of this inclusive set of chimeras indicate that the first two cytoplasmic loops of the cardiac sodium channel that join domain I to II (loop A), and domain II to III (loop B), are both necessary, and together are sufficient to produce a papain-induced hyperpolarising shift in the voltage at which channels activate. When both loops are present (wild-type hH1 and the chimera hSkM1AB), V(a) for the channel shifts in the hyperpolarised direction by > 20 mV with papain treatment. When the analogous hSkM1 loops are present (wild-type hSkM1 and the chimera hH1AB), V(a) for the channel is not sensitive to treatment with papain. For channels that contain only one of the two hH1 loops, the effect of papain on V(a) is intermediary. 5. Experiments performed in the absence of papain showed that the activation voltages of the double loop chimeras, hSkM1AB and hH1AB, were shifted significantly from V(a) for hSkM1 and V(a) for hH1, respectively, indicating that these loops directly alter channel activation voltage. The resulting shifts in V(a) were in opposing directions, suggesting that cytoplasmic control of activation voltage is isoform specific. V(a) for hSkM1AB was about 20 mV more depolarised than V(a) for hSkM1, and V(a) for hH1AB was about 9 mV more negative than V(a) for hH1. 6. These data are the first to indicate isoform-specific cytoplasmic regions of the voltage-gated sodium channel that directly and differently alter the voltage of channel activation.