Author(s): Agustin Garcia-Caballero1 , Maria A. Gandini1 , Shuo Huang1 , Lina Chen1 , Ivana A. Souza1 , Yan L. Dang2 , M. Jackson Stutts2 and Gerald W. Zamponi1
The Epithelial Sodium Channel (ENaC) is a key regulator of sodium absorption in a variety of tissues. It fine tunes sodium in the nephron and the colonic epithelia  where it helps to maintain total body salt and volume homeostasis. In the pulmonary airway epithelia ENaC regulates the composition and depth of the airway surface liquid to maintain mucociliary clearance [2, 3]. Malfunction of ENaC in these tissues results in various cardiovascular  and lung diseases, such as cystic fibrosis . ENaC typically consists of three subunits ([alpha], [beta], and [gamma]) which when co-assembled form a sodium selective pore . Each subunit is composed of two transmembrane domains with an amino-terminal (N-terminal) cytosolic domain, a large extracellular loop and a cytosolic carboxyl-terminus (C-terminus) . Gain or loss of function mutations in all ENaC subunits result in disorders such as hypertension or Liddle's Syndrome and pseudohypoaldosteronism [4, 7]. Importantly, ENaC is inhibited by the Cystic Fibrosis transmembrane conductance regulator (CFTR) via channel / channel interactions . ENaC can also interact with the thiazide-sensitive sodium chloride cotransporter (NCC) in the kidney , suggesting that these channels can readily interact with other membrane proteins. ENaC expression is not limited to non-neuronal tissues as evident from reported expression in mechanosensory neurons , dorsal root ganglia (DRG)  and brain. However, the function of ENaC channels in neurons has not been well described.
Certain types of sensory neurons express Cav3.2 calcium channels [12, 13] These channels belong to the family of low-voltage gated T-type calcium channels . Their biophysical properties, such as low voltage thresholds for activation and inactivation, fast inactivation and rebound bursting, are important for neuronal excitability in both the central and peripheral nervous systems [14, 15]. Cav3.2 calcium channels are also expressed in the thalamus  where they play an important role in epilepsy [16-18]. Importantly, Cav3.2 calcium channels can form channel complexes with members of the potassium channel family such as Kv4, KCa1.1, and KCa3.1 to regulate neuronal activity [19-21].
Because of the overlap in ENaC and Cav3.2 channel expression in sensory neurons, and the reported inhibitory effect of intracellular calcium on the open probability of ENaC and on ENaC expression [22-24], we tested whether Cav3.2 channels may form a protein complex with ENaC. We find that specific ENaC channel subunits interact with Cav3.2 channels and reciprocally regulate each other's membrane expression.
Cell culture and transfection
Human embryonic kidney tsA-201 cells were cultured as described previously . Cells were transfected with calcium phosphate and used for biochemical and electrophysiological analysis 48-72 h post-transfection. Mouse CAD cells were cultured as described previously . Cells were transfected with lipofectamine 2000 and used for biochemical assays 48-72 h post-transfection.
cDNAs encoding rat [alpha]-, [beta]-, and [gamma]-ENaC with HA-N-terminal (HA-NT) epitope tags were used. WT and mutant constructs ([beta]-ENaC: K4R/ K5R/...