Paramyotonia Congenita Mutations Reveal Different Roles for Segments $3 and $4 of Domain D4 in hSkM1 Sodium Channel Gating

Mutations in the gene encoding the voltage-gated sodium channel of skeletal muscle (SkM1) have been identified in a group of autosomal dominant diseases, characterized by abnormalities of the sarcolemmal excitability, that include paramyotonia congenita (PC) and hyperkalemic periodic paralysis (HYPP). We previously reported that PC mutations cause in common a slowing of inactivation in the human SkM1 sodium channel. In this investigation, we examined the molecular mechanisms responsible for the effects of L1433R, located in D4/$3, on channel gating by creating a series of additional mutations at the 1433 site. Unlike the R1448C mutation, found in D4/$4, which produces its effects largely due to the loss of the positive charge, change of the hydropathy of the side chain rather than charge is the primary factor mediating the effects of L1433R. These two mutations also differ in their effects on recovery from inactivation, conditioned inactivation, and steady state inactivation of the hSkM1 channels. We constructed a double mutation containing both L1433R and R1448C. The double mutation closely resembled R1448C with respect to alterations in the kinetics of inactivation during depolarization and voltage dependence, but was indistinguishable from L1433R in the kinetics of recovery from inactivation and steady state inactivation. No additive effects were seen, suggesting that these two segments interact during gating. In addition, we found that these mutations have different effects on the delay of recovery from inactivation and the kinetics of the tail currents, raising a question whether this delay is a reflection of the deactivation process. These results suggest that the $3 and $4 segments play distinct roles in different processes of hSkM1 channel gating: D4/$4 is critical for the deactivation and inactivation of the open channel while D4/$3 has a dominant role in the recovery of inactivated channels. However, these two segments interact during the entry to, and exit from, inactivation states. I N T R O D U C T I O N Abnormalities in the skeletal muscle voltage-gated sodium channel (SkM1), a protein essential for generation of the action potential in sarcolemma, can have important consequences for muscle contraction (Barchi, 1995). Such consequences are exemplified by a group of autosomal dominant hereditary muscle diseases, including paramyotonia congenita (PC) and hyperkalemic periodic paralysis (HYPP), in which sympAddress correspondence to R. L. Barchi, Institute of Neurological Sciences, 218 Stemmler Hall, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6074. 183 toms can be attributed to either hyperexcitability or hypoexcitability of the sarcolemma. The relationship of these diseases to the sodium channel in skeletal muscle was first suggested by the identification of a tetrodotoxin (TTX)-sensitive, noninactivating inward current in muscle fibers obtained from patients having PC or HYPP. A causal relationship between defects in sodium channels and these diseases was established when genetic linkage analysis showed both HYPP and PC to be allelic disorders of the human adult skeletal muscle sodium channel 0~ subunit (hSkM1) gene (SCN4A). So far 16 point mutations, widely distributed throughout the primary sequence of domains 2 to 4 of the hSkM1 sodium channel, have been reported in PC and HYPP patients (Barchi, 1995;Ji et al., 1995). j. GEN. PHYSIOL. 9 The Rockefeller University Press 9 0022-1295/96/02/183/12 $2.00 Volume 107 February 1996 183-194 on Jne 1, 2017 D ow nladed fom Published February 1, 1996