Programme Contents

Molecular and biophysical properties of smooth muscle-type voltage-gated Na⁺ channels

It is well-known that activation of voltage-dependent Ca²⁺ channels (Ca_V) and voltage-gated Na⁺ channels (Na_V) is involved in the generation of action potentials in various types of excitable cells. Although voltage-gated Na⁺ currents (I_Na) fail to be recorded in the vast majority of smooth muscle tissues, there are several reports regarding the existence of I_Na in vascular and visceral smooth muscles, suggesting that I_Na are involved in the generation of action potentials. Na_V appear to be selectively expressed in some, but not all smooth muscle, thereby questioning their significance in the physiology of the tissues. Furthermore, little attention has been given to the molecular properties of TTX-sensitive I_Na in smooth muscles.

Recent studies have revealed that Na_V consist of three subunits (expressed as a trimer): namely, an α subunit (260 kDa) which forms the core protein of the channel (possessing the TTX-binding sites) and two β subunits (30-40 kDa) which modify the channel function as an auxiliary subunit. To date, eleven isoforms of genes (Scn1a-11a) encoding TTX-sensitive and TTX-insensitive Na_V have been identified within a single family of Na_V, Na_V1.X and four isoforms of genes (Scn1b-4b) encoding β subunits have been also detected.

The electrophysiological and pharmacological properties of Na_V in murine vas deferens smooth muscle cells were investigated using patch-clamp techniques. In whole-cell configuration, a fast, transient inward current was evoked in the presence of Cd²⁺, and was abolished by TTX (K_d = 11.2 nM), mibefradil (K_d = 3.3 μM) and external replacement of Na⁺ with monovalent cations (TEA⁺, Tris⁺ and NMDG⁺). The fast transient inward current was enhanced by veratridine, an activator of voltage-gated Na⁺ channels, suggesting that the fast transient inward current was a TTX-sensitive I_Na. The values for half-maximal (V_half) inactivation and activation of I_Na were -46.3 mV and -26.0 mV respectively. The molecular identity of the TTX-sensitive pore-forming subunits was revealed using RT-PCR analysis, in situ hybridization and immunohistochemistry. RT-PCR analysis revealed the expression of Scn1a, 2a and 8a transcripts, whilst Scn1b was only detected. The Scn8a transcript and the α subunit protein of Na_V1.6 were detected in smooth muscle layers. Furthermore, using Na_V1.6-null mice (Na_V1.6^–/–) lacking the expression of the Na⁺ channel gene, Scn8a, I_Na were not detected in dispersed smooth muscle cells from the vas deferens, whilst TTX-sensitive I_Na were recorded in their wild-type (Na_V1.6^+/+) littermates. This study demonstrates that the molecular identity of the Na_V responsible for the TTX-sensitive I_Na in murine vas deferens myocytes is primarily Na_V1.6. (Zhu et al., 2008), co-expressed with β1 subunits.

Zhu HL, Aishima M, Morinaga H, Wassall RD, Shibata A, Iwasa K, Nomura M, Nagao M, Sueishi K, Cunnane TC, Teramoto N (2008) Biophysical Journal 94, 3340-3351.