A potential role for sialic acid in the voltage-dependent gating of rat skeletal muscle sodium channels (rSkM1) was investigated using Chinese hamster ovary (CHO) cells stably transfected with rSkM1. Changes in the voltage dependence of channel gating were observed after enzymatic (neuraminidase) removal of sialic acid from cells expressing rSkM1 and through the expression of rSkM1 in a sialylation-deficient cell line (lec2). The steady-state half-activation voltages (Va) of channels under each condition of reduced sialylation were ∼10 mV more depolarized than control channels. The voltage dependence of the time constants of channel activation and inactivation were also shifted in the same direction and by a similar magnitude. In addition, recombinant deletion of likely glycosylation sites from the rSkM1 sequence resulted in mutant channels that gated at voltages up to 10 mV more positive than wild-type channels. Thus three independent means of reducing channel sialylation show very similar effects on the voltage dependence of channel gating. Finally, steady-state activation voltages for channels subjected to reduced sialylation conditions were much less sensitive to the effects of external calcium than those measured under control conditions, indicating that sialic acid directly contributes to the negative surface potential. These results are consistent with an electrostatic mechanism by which external, negatively charged sialic acid residues on rSkM1 alter the electric field sensed by channel gating elements.

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. Because channel gating is dependent directly on the membrane potential, anything that alters this potential will affect gating. For example, external calcium alters the voltage dependence of channel activation such that as external calcium is increased, a greater depolarization is required in order to achieve the same degree of channel opening (Frankenhauser & Hodgkin, 1957; Bennett et al. 1997; Hille, 2001). The surface charge hypothesis predicts that the surface of the membrane near the channel has fixed negative charges that alter the electric field sensed by the gating mechanism of the channel. These negative charges are formally equivalent to internal fixed positive charges that will depolarize the membrane and thereby move the transmembrane potential closer to the threshold for channel opening. Calcium may act to neutralize the effects of these external negative charges, either through direct interaction with these charges or by a screening mechanism, effectively hyperpolarizing the membrane. Therefore, depolarizations sufficient to activate channels in low calcium are no longer adequate in elevated calcium.Patients with misregulated plasma calcium levels (hypoand hypercalcaemia) show symptoms consistent with direct effects of calcium on sodium channel gating The isoform specific role of sialic acid in human voltage-gated sodium channel gating was investigated through expression and chimeric analysis of two human isoforms, Na v1.4 (hSkM1), and Na v1.5 (hH1) in Chinese hamster ovary (CHO) cell lines. Immunoblot analyses indicate that both hSkM1 and hH1 are glycosylated and that hSkM1 is more glycosylated than hH1. Four sets of voltage-dependent parameters, the voltage of half-activation (V a ), the voltage of half-inactivation (V i ), the time constants for fast inactivation (t h ), and the time constants for recovery from inactivation (t rec ), were measured for hSkM1 and hH1 expressed in two CHO cell lines, Pro5 and Lec2, to determine the effect of changing sialylation on channel gating under conditions of full (Pro5) or reduced (Lec2) sialylation. For all parameters measured, hSkM1 gating showed a consistent 11_15 mV depolarizing shift under conditions of reduced sialylation, while hH1 showed no significant change in any gating parameter. Shifts in channel V a with changing external [Ca 2+ ] indicated that sialylation of hSkM1, but not hH1, directly contributes to a negative surface potential. Functional analysis of two chimeras, hSkM1P1 and hH1P1, indicated that the responsible sialic acids are localized to the hSkM1 S5-S6 loop of domain I. When hSkM1 IS5-S6 was replaced by t...

Voltage-gated sodium channels (Na v ) are responsible for initiation and propagation of nerve, skeletal muscle, and cardiac action potentials. Na v are composed of a pore-forming ␣ subunit and often one to several modulating ␤ subunits. Previous work showed that terminal sialic acid residues attached to ␣ subunits affect channel gating. Here we show that the fully sialylated ␤ 1 subunit induces a uniform, hyperpolarizing shift in steady state and kinetic gating of the cardiac and two neuronal ␣ subunit isoforms. Under conditions of reduced sialylation, the ␤ 1 -induced gating effect was eliminated. Consistent with this, mutation of ␤ 1 N-glycosylation sites abolished all effects of ␤ 1 on channel gating. Data also suggest an interaction between the cis effect of ␣ sialic acids and the trans effect of ␤ 1 sialic acids on channel gating. Thus, ␤ 1 sialic acids had no effect on the gating of the heavily glycosylated skeletal muscle ␣ subunit. However, when glycosylation of the skeletal muscle ␣ subunit was reduced through chimeragenesis such that ␣ sialic acids did not impact gating, ␤ 1 sialic acids caused a significant hyperpolarizing shift in channel gating. Together, the data indicate that ␤ 1 N-linked sialic acids can modulate Na v gating through an apparent saturating electrostatic mechanism. A model is proposed in which a spectrum of differentially sialylated Na v can directly modulate channel gating, thereby impacting cardiac, skeletal muscle, and neuronal excitability.