Comparison of slow inactivation in human heart and rat skeletal muscle Na+channel chimaeras

1. Voltage-gated Na+ channels undergo two types of inactivation in response to depolarization. One type, fast inactivation, occurs with a time scale o f milliseconds. The other, slow inactivation, occurs over seconds to minute s. In addition, these two processes appear to be distinct at the molecular level. However, the molecular mechanism of Na+ channel slow inactivation is unknown. 2. We used patch clamp techniques to study slow inactivation, activation an d fast inactivation in a-subunit cDNA clones for wild-type human heart Nachannels (hH1) and rat skeletal muscle Na+ channels (mu 1) transiently expr essed in human embryonic kidney (HEK) cells. Our experiments showed that th e Na+ channel slow inactivation phenotype (development, steady state and re covery) differed dramatically between hH1 and mu 1. Slow inactivation in mu 1 had a faster onset, a steeper voltage dependence, and was more complete compared with hH1. In addition, recovery from slow inactivation was much sl ower for mu 1 than for hH1. Activation and fast inactivation kinetics were also different in hH1 and mu 1. In hill, fast inactivation was slower and V -1/2 values of activation and steady-state fast inactivation (h(infinity)) were more negative than in mu 1. 3. To better understand the molecular basis of Na+ channel slow inactivatio n, Na+ channel chimaeras were constructed with domains from hH1 and mu 1. T he slow inactivation phenotype in the chimaeras (domains denoted by subscri pts) mu 1((1))hH1((2,3,4)), mu 1((1,2))hH1((3,4)), and mu 1((1,2,3))hH1((4) ) was intermediate compared with that of wild-type. However, the chimaera m u 1((1))hH1((2,3,4)) was more like mild-type hH1, while the chimaeras mu 1( (2,3))hH1((3,4)) and mu 1((1,2,3))hH1((4)), were more similar to wild-type mu 1. In the chimaeras, activation resembled that of mu 1, fast inactivatio n resembled that of hH1, and steady-state fast inactivation fell between th at of hH1 and mu 1. 4. The data demonstrate that all four domains can modulate the Na+ channel slow inactivation phenotype. However, domains D1 and D2 may play a more pro minent role in determining Nat channel slow inactivation phenotype than D3 and D4. The results also support previous conclusions that D3 and D4 land t he D3-D4 linker) play an important role in Naf channel fast inactivation, a nd that activation may require non-equivalent contributions from all four d omains.