It is well-known that activation of voltage-dependent Ca2+ channels (CaV) and voltage-gated Na+ channels (NaV) is involved in the generation of action potentials in various types of excitable cells. Although voltage-gated Na+ currents (INa) fail to be recorded in the vast majority of smooth muscle tissues, there are several reports regarding the existence of INa in vascular and visceral smooth muscles, suggesting that INa are involved in the generation of action potentials. NaV 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 INa in smooth muscles.
Recent studies have revealed that NaV 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 NaV have been identified within a single family of NaV, NaV1.X and four isoforms of genes (Scn1b-4b) encoding β subunits have been also detected.
The electrophysiological and pharmacological properties of NaV 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 (Kd = 11.2 nM), mibefradil (Kd = 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 INa. The values for half-maximal (Vhalf) inactivation and activation of INa 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 NaV1.6 were detected in smooth muscle layers. Furthermore, using NaV1.6-null mice (NaV1.6–/–) lacking the expression of the Na+ channel gene, Scn8a, INa were not detected in dispersed smooth muscle cells from the vas deferens, whilst TTX-sensitive INa were recorded in their wild-type (NaV1.6+/+) littermates. This study demonstrates that the molecular identity of the NaV responsible for the TTX-sensitive INa in murine vas deferens myocytes is primarily NaV1.6. (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.