Secondary structure of the human cardiac Na+ channel C terminus: evidence for a role of helical structures in modulation of channel inactivation.
Little is known about the structure of the C terminus of the human cardiac voltage-gated sodium channel alpha subunit (SCN5A), but disease-linked mutations within this 244-amino acid intracellular region of the channel have marked effects on channel inactivation. Here we report a structural analysis of the C-terminal tail of the cardiac Na(+) channel that sheds new light on mechanisms that control its inactivation gating. Homology modeling of the SCN5A C terminus predicts predominant alpha-helical structure (six helices) in the proximal half of this intracellular tail but little structure in the distal half. Circular dichroism of isolated and purified C terminus supports this prediction. Whole cell and single channel patch clamp recordings of wild type and mutant alpha subunits co-expressed with the hbeta(1) subunit in HEK 293 cells indicate that truncation of the distal, nonstructured, C terminus (L1921stop mutant) reduces current density but does not affect channel gating (n = 6). In contrast, truncation of the sixth helix containing a concentration of positively charged residues along with the distal C terminus (S1885stop mutant) also reduces current density but, in addition, has profound and selective effects on inactivation (no effect on activation). Channel availability is shifted (-11 +/- 0.6 mV), and there is a 10-fold increase in the percentage of channels that burst (fail to inactivate) during prolonged depolarization (0.025% S1885stop (n = 7) versus 0.0028% wild type (n = 9), p < 0.005). These results suggest that the charged structured region of the SCN5A C terminus plays a major role in channel inactivation, stabilizing the inactivated state.
Cormier
Joseph W
JW
Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
Rivolta
Ilaria
I
Tateyama
Michihiro
M
Yang
An-Suei
AS
Kass
Robert S
RS
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006801:Humans; D008969:Molecular Sequence Data; D062554:NAV1.5 Voltage-Gated Sodium Channel; D017433:Protein Structure, Secondary; D015222:Sodium Channels / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology
D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; D015222:Sodium Channels