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

Voltage-gated sodium channels are responsible for the initiation and propagation of nerve, skeletal muscle and cardiac action potentials. The orchestrated activation and inactivation gating of sodium channels is vital to normal neuronal signalling, skeletal muscle contraction and normal heart rhythms. Even small syncopations from this normal gating rhythm may alter cellular excitability and whole animal physiology significantly. Several genetic sodium channel diseases have been described, with multiple mutations leading to similar disease phenotypes. Diseases such as paramyotonia congenita (PMC), hyperkalaemic periodic paralysis (HYPP) and long QT syndrome (LQTS), are examples of well studied genetic diseases of skeletal muscle and cardiac voltage-gated sodium channels (see e.g. Cannon et al. 1991; Ptacek et al. 1991, 1992; Cummins et al. 1993; Chahine et al. 1994; Bennett et al. 1995; Dumaine et al. 1996; Fan et al. 1996; Hayward et al. 1996; Ji et al. 1996; Lawrence et al. 1996; Lerche et al. 1996; Wang et al. 1996b; Nagatomo et al. 1998). These mutant channels typically show small ‘persistent’ sodium currents when studied in vitro. Channel inactivation is altered through mutation, resulting in a small percentage of channels residing in the active state longer than the wild-type channel. The result is a small increase in inward current, causing a slight depolarisation of the membrane. While channel function is only minimally affected, the whole animal phenotype is significantly altered. Patients present with such symptoms as muscle paralysis or cardiac arrhythmias caused by the slight redistribution of channels from one functional state to another and a slight change in resting membrane potential. Like many other ion channels, voltage-gated sodium channels are molecularly diverse, with different channel isoforms expressed specifically by different cell types or at different times throughout development (for reviews see Kallen et al. 1993; Catterall, 1995; Jan & Jan, 1997; Marban et al. 1998; Goldin, 2001). Often several isoforms are expressed within a single cell. While most efforts have focused on the functional role of conserved structures or the impact of natural mutations on channel function, recent work has studied functional differences between isoforms of the same ion channel species. Comparisons of steady state activation and inactivation voltages, rates of fast and slow inactivation and recovery from inactivation, as well as affinities and actions of toxins and anaesthetics in neuronal, cardiac and skeletal muscle sodium channel isoforms have been described (see e.g. Chen et al. 1992; Nuss et al. 1995a, b; Rehberg et al. 1995; Chahine et al. 1996; Makita et al. 1996; Wang et al. 1996a; Bendahhou et al. 1997; Dib-Hajj et al. 1997; Wright et al. 1997; Deschenes et al. 1998; O'Leary, 1998; Bennett, 1999; O'Reilly et al. 1999; Sheets & Hanck, 1999; Villin et al. 1999; Wei et al. 1999). Generally, these studies were designed to help understand how relatively subtle functional differences between sodium channel isoforms are responsible for the observed physiology in excitable cells. A recent paper from this laboratory (Bennett, 1999), revealed a novel functional difference between two sodium channel isoforms, adult rat skeletal muscle sodium channel (rSkM1) and human heart sodium channel 1 (hH1). While internal papain treatment removed both rSkM1 and hH1 fast inactivation, the half-activation voltage (Va) for hH1 was shifted dramatically in the hyperpolarised direction with no significant impact on Va for rSkM1. These data imply that cytoplasmic structural differences between cardiac and skeletal muscle sodium channel isoforms exist that affect channel activation differently. In this study, attempts were made to determine which specific cytoplasmic regions of the cardiac sodium channel are responsible for this papain-induced shift in Va. Studies of the adult human skeletal muscle isoform (hSkM1) indicate that the channel behaves similarly to its rat orthologue. That is, the activation voltage of hSkM1 is unaffected by treatment with papain. Functional comparison of hH1/hSkM1 chimeras indicates that the first two cytoplasmic loops of the cardiac sodium channel joining domain I to II (loop A) and domain II to III (loop B), are both necessary and together sufficient to produce a significant papain-induced, hyperpolarising shift in the voltage at which the cardiac channel activates. In addition, activation voltages of the chimeras differ from Va for wild-type in the absence of papain, with hSkM1 loops A and B imposing a hyperpolarising effect on Va for the channel, while loops A and B of hH1 shift Va for the channel in the depolarising direction.