Abstract-Amino acid sequence variations in SCN5A are known to affect function of wild-type channels and also those with coexisting mutations; therefore, it is important to know the exact sequence and function of channels most commonly present in human myocardium. SCN5A was analyzed in control panels of human alleles, demonstrating that the existing clones (hH1, hH1a, hH1b) each contained a rare variant and thus none represented the common sequence. Confirming prior work, the H558R polymorphism was present in Ϸ30% of subjects. Quantitative mRNA analysis from human hearts showed that a shorter 2015 amino acid splice variant lacking glutamine at position 1077 (Q1077del) made up 65% of the transcript in every heart examined. Age, sex, race, or structural heart disease did not affect this proportion of Q1077del. Estimated population frequencies for the four common variants were 25% SCN5A, 10% [H558R], 45% [Q1077del], and 20% [H558R;Q1077del], where the reference sequence SCN5A is GenBank AC137587. When expressed in HEK-293 cells, these common variants had a more positive mid-point of the voltage dependence of inactivation than the standard clone hH1. Also, channels containing Q1077 expressed smaller currents. When H558R was present with Q1077 ([H558R]), current expression was profoundly reduced despite normal trafficking to the cell surface. Thus, four variant sequences for SCN5A are commonly present in human myocardium and they exhibit functional differences among themselves and with the previous standard clone. SCN5A encodes the voltage-dependent sodium channel ␣-subunit protein SCN5A, also called hNa v 1.5, 1 found predominantly in human heart muscle. This channel is responsible for large peak inward sodium current (I Na ) that underlies excitability and conduction in working myocardium (atrial and ventricular cells) and special conduction tissue (Purkinje cells and others), and also for late I Na that influences repolarization and refractoriness. Three complete cDNA clones for this channel hH1, 2 hH1a, 3 and hH1b 4 differ in amino acid sequence in 5 of the 2016 positions (Table 1). In addition, these three clones differ from the deduced amino acid sequence for SCN5A obtained from the two human genome databases: Celera and the International Human Genome Sequencing Collaboration (IHGSC). Before the present study, it was not clear whether or not these differences are present in human population as common variants. From previous studies, we know that dramatic differences in current expression can be found when arrhythmia mutations are expressed in different background clones. 4 This study was designed to answer the questions: What is the common background sequence for SCN5A? Do common variations affect channel function? Does it matter which Na ϩ channel clone (ie, background sequence) is used for functional studies of wild-type and mutated channels? Materials and MethodsProtocols used in this investigation are more fully described in the expanded Materials and Methods section in the online data supplement available...

The gating ring of cyclic nucleotide-modulated channels is proposed to be either a two-fold symmetric dimer of dimers or a four-fold symmetric tetramer based on high-resolution structure data of soluble cyclic nucleotide-binding domains and functional data on intact channels. We addressed this controversy by obtaining structural data on an intact, full-length, cyclic nucleotide-modulated potassium channel, MloK1, from Mesorhizobium loti, which also features a putative voltage-sensor. We present here the 3D single-particle structure by transmission electron microscopy and the projection map of membrane-reconstituted 2D crystals of MloK1 in the presence of cAMP. Our data show a four-fold symmetric arrangement of the CNBDs, separated by discrete gaps. A homology model for full-length MloK1 suggests a vertical orientation for the CNBDs. The 2D crystal packing in the membrane-embedded state is compatible with the S1-S4 domains in the vertical "up" state.

No abstract

No abstract