The immunoglobulin domain of the sodium channel ß3 subunit contains a surface-localized disulfide bond that is required for homophilic binding.

The ß subunits of voltage-gated sodium (Na(v)) channels possess an extracellular immunoglobulin (Ig) domain that is related to the L1 family of cell-adhesion molecules (CAMs). Here we show that in HEK293 cells, secretion of the free Ig domain of the ß3 subunit is reduced significantly when it is coexpressed with the full-length ß3 and ß1 subunits but not with the ß2 subunit. Using immunoprecipitation, we show that the ß3 subunit can mediate trans homophilic-binding via its Ig domain and that the ß3-Ig domain can associate heterophilically with the ß1 subunit. Evolutionary tracing analysis and structural modeling identified a cluster of surface-localized amino acids fully conserved between the Ig domains of all known ß3 and ß1 sequences. A notable feature of this conserved surface cluster is the presence of two adjacent cysteine residues that previously we have suggested may form a disulfide bond. We now confirm the presence of the disulfide bond in ß3 using mass spectrometry, and we show that its integrity is essential for the association of the full-length, membrane-anchored ß3 subunit with itself. However, selective reduction of this surface disulfide bond did not inhibit homophilic binding of the purified ß3-Ig domain in free solution. Hence, the disulfide bond itself is unlikely to be part of the homophilic binding site. Rather, we suggest that its integrity ensures the Ig domain of the membrane-tethered ß3 subunit adopts the correct orientation for productive association to occur in vivo.

The sodium channel {beta}3-subunit induces multiphasic gating in NaV1.3 and affects fast inactivation via distinct intracellular regions.

Electrical excitability in neurons depends on the activity of membrane-bound voltage gated sodium channels (Na(v)) that are assembled from an ion conducting α-subunit and often auxiliary ß-subunits. The α-subunit isoform Na(v)1.3 occurs in peripheral neurons together with the Na(v) ß3-subunit, both of which are coordinately up-regulated in rat dorsal root ganglion neurons after nerve injury. Here we examine the effect of the ß3-subunit on the gating behavior of Na(v)1.3 using whole cell patch clamp electrophysiology in HEK-293 cells. We show that ß3 depolarizes the voltage sensitivity of Na(v)1.3 activation and inactivation and induces biphasic components of the inactivation curve. We detect both a fast and a novel slower component of inactivation, and we show that the ß3-subunit increases the fraction of channels inactivating by the slower component. Using CD and NMR spectroscopy, we report the first structural analysis of the intracellular domain of any Na(v) ß-subunit. We infer the presence of a region within the ß3-subunit intracellular domain that has a propensity to form a short amphipathic α-helix followed by a structurally disordered sequence, and we demonstrate a role for both of these regions in the selective stabilization of fast inactivation. The complex gating behavior induced by ß3 may contribute to the known hyperexcitability of peripheral neurons under those physiological conditions where expression of ß3 and Na(v)1.3 are both enhanced.

The importance of serine 161 in the sodium channel beta3 subunit for modulation of Na(V)1.2 gating.

Voltage-gated sodium (Na) channels contribute to the regulation of cellular excitability due to their role in the generation and propagation of action potentials. They are composed of a pore-forming alpha subunit and are modulated by at least two of four distinct beta subunits (beta1-4). Recent studies have implicated a role for the intracellular domain of beta subunits in modulating Na channel gating and trafficking. In beta3, the intracellular domain contains a serine residue at position 161 that is replaced by an alanine in beta1. In this study, we have probed the functional importance of beta3S161 for modulating Na channel gating. Wild-type beta3 and point mutations beta3S161A or beta3S161E were individually co-expressed in HEK 293 cells stably expressing human Na(v)1.2. WTbeta3 expression increased Na current density, shifted steady-state inactivation in a depolarized direction, and accelerated the kinetics of recovery from inactivation of the Na current. Analogous effects were observed with beta3S161E co-expression. In contrast, beta3S161A abolished the shifts in steady-state inactivation and recovery from inactivation of the Na current, but did increase Na current density. Immunocytochemistry and Western blot experiments demonstrate membrane expression of WTbeta3, beta3S161E, and beta3S161A, suggesting that the differences in Na channel gating were not due to disruptions in beta subunit trafficking. These studies suggest that modification of beta3S161 may be important in modulating Na-channel gating.

A new look at sodium channel ß subunits.

Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated ß subunits. The ß subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human ß3 and ß4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that ß3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some ß subunits.

Trafficking and cellular distribution of voltage-gated sodium channels.

Electrical excitability in cells such as neurons and myocytes depends not only upon the expression of voltage-gated sodium channels but also on their correct targeting within the plasma membrane. Placing sodium channels within a broader cell biological context is beginning to shed new light on a variety of important questions such as the integration of neuronal signaling. Mutations that affect sodium channel trafficking have been shown to underlie several life-threatening conditions including cardiac arrhythmias, revealing an important clinical context to these studies.

Tissue distribution profiles of the human TRPM cation channel family.

Eight members of the TRP-melastatin (TRPM) subfamily have been identified, whose physiological functions and distribution are poorly characterized. Although tissue expression and distribution patterns have been reported for individual TRPM channels, comparisons between individual studies are not possible because of variations in analysis techniques and tissue selection. We report here a comparative analysis of the expression patterns of all of the human TRPM channels in selected peripheral tissues and the central nervous system (CNS) using two distinct but complimentary approaches: TaqMan and SYBR Green real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). These techniques generated comparative distribution profiles and demonstrated tissue-specific co-expression of TRPM mRNA species, indicating significant potential for the formation of heteromeric channels. TRPM channels 2, 4, 5, 6, and 7 in contrast to 1, 3, and 8 are widely distributed in the CNS and periphery. The tissues demonstrating highest expression for individual family members were brain (TRPM1), brain and bone marrow (TRPM2), brain and pituitary (TRPM3), intestine and prostate (TRPM4), intestine, pancreas, and prostate (TRPM5), intestine and brain (TRPM6), heart, pituitary, bone, and adipose tissue (TRPM7), and prostate and liver (TRPM8). The data reported here will guide the elucidation of TRPM channel physiological functions.