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Molecular and Biophysical Properties of Voltage-Gated Na⁺ Channels in Murine Vas Deferens

Hai-Lei Zhu ^*, Manami Aishima ^*, Hidetaka Morinaga , Richard D. Wassall , Atsushi Shibata ^*, Kazuomi Iwasa ^*, Masatoshi Nomura , Masaya Nagao , Katsuo Sueishi ^¶, Thomas C. Cunnane  and Noriyoshi Teramoto ^*

^* Department of Pharmacology, Department of Medicine and Bioregulatory Science, and ^¶ Division of Pathophysiological and Experimental Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Department of Pharmacology, University of Oxford, Oxford, United Kingdom; and Laboratory of Biosignals and Response, Graduate School of Biostudies, Kyoto University, Kyoto, Japan

Correspondence: Address reprint requests to Noriyoshi Teramoto, Tel.: 81-92-642-6077; E-mail: noritera{at}med.kyushu-u.ac.jp.

The biological and molecular properties of tetrodotoxin (TTX)-sensitive voltage-gated Na⁺ currents (I_Na) in murine vas deferens myocytes were investigated using patch-clamp techniques and molecular biological analyses. 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. RT-PCR analysis revealed the expression of Scn1a, 2a, and 8a transcripts. The Scn8a transcript and the -subunit protein of Na_V1.6 were detected in smooth muscle layers. 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, while TTX-sensitive I_Na were recorded in their wild-type (Na_V1.6^+/+) littermates. This study demonstrates that the molecular identity of the voltage-gated Na⁺ channels responsible for the TTX-sensitive I_Na in murine vas deferens myocytes is primarily Na_V1.6.