Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis

Abstract

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca²⁺-activated Cl⁻ channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential −60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl⁻ concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα⁺, ANO1⁺ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca²⁺ release from intracellular Ca²⁺ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca²⁺ channels are activated to develop SPCs.