Ano1 mediates pressure-sensitive contraction frequency changes in mouse lymphatic collecting vessels

Lymphatic collecting vessels exhibit spontaneous contractions with a pressure-dependent contraction frequency. The initiation of contraction has been proposed to be mediated by the activity of a Ca²⁺-activated Cl^- channel (CaCC). Here, we show that the canonical CaCC Anoctamin 1 (Ano1, TMEM16a) plays an important role in lymphatic smooth muscle pacemaking. We find that isolated murine lymphatic muscle cells express Ano1, and demonstrate functional CaCC currents that can be inhibited by the Ano1 inhibitor benzbromarone. These currents are absent in lymphatic muscle cells from Cre transgenic mouse lines targeted for Ano1 genetic deletion in smooth muscle. We additionally show that loss of functional Ano1 in murine inguinal-axillary lymphatic vessels, whether through genetic manipulation or pharmacological inhibition, results in an impairment of the pressure-frequency relationship that is attributable to a hyperpolarized resting membrane potential and a significantly depressed diastolic depolarization rate preceding each action potential. These changes are accompanied by alterations in action potential shape and duration, and a reduced duration but increased amplitude of the action potential-induced global "Ca²⁺ flashes" that precede lymphatic contractions. These findings suggest that an excitatory Cl^- current provided by Ano1 is critical for mediating the pressure-sensitive contractile response and is a major component of the murine lymphatic action potential.