The effect of Na⁺ removal was studied using a patch clamp technique in single PAMs at room temperature. Male Wistar rats (225-300 g) were humanely killed and PAMs were isolated from small pulmonary arteries (〈400 μM external diameter) using collagenase and papain (Smirnov et al., 2002). Voltage-dependent K⁺ (Kv) current (I_Kv) was isolated using 1 μM paxilline and 10 μM glybenclamide (to eliminate BK_Ca and K_ATP respectively) in the external physiological saline solution (PSS) containing (mM): 130 NaCl, 5 KCl, 1.2 MgCl₂, 1.5 CaCl₂, 10 HEPES, 10 glucose, pH=7.2. Cells were dialysed with a pipette solution of composition (mM): 130 KCl, 0.5 MgCl₂, 0.5 CaCl₂, 10 HEPES, 10 EGTA, pH=7.2, unless mentioned otherwise. I_Kv was investigated using a 2 s voltage ramp applied every 20 s between -100 to 100 mV from a holding potential of -80 mV. Removal of extracellular sodium (Na⁺-free PSS, equimolar substitution with N-methyl-D-glucamine) augmented I_Kv from -60 to 10 mV causing a leftward shift in I_Kv activation. Changes in the I_Kv activation were described using the Boltzmann function and showed a significant decrease in the I_Kv half-activation potential (V_a) from 6.5±2.4 mV (PSS) to -16±3 mV (Na⁺-free PSS) (n=19, p〈0.0001, paired Students t test), yielding a mean relative shift in the I_Kv activation (ΔV_a) of -22.4±3.1 mV (n=19). Values are given as mean±s.e.m and statistically compared using unpaired t test, unless stated otherwise. Incubation of PAMs with 100 μM amiloride, an inhibitor of the Na⁺-Mg²⁺ exchanger, for 6 min caused a leftward shift (ΔV_a =-9.5±2.4 mV, n=9, p〈0.015) mimicking that observed in the Na⁺-free PSS. Subsequent removal of Na⁺ in the presence of amiloride had only a small additional effect on the I_Kv activation (ΔV_a =-5.6±3.3 mV, n=8, p〈0.006). Similarly, increased pipette MgCl₂ to 10 mM produced a leftward shift (V_a =-7.3±3.6 mV, n=8, p〈0.004) and, though not significantly, diminished the effect of Na⁺-free PSS (ΔV_a =-16.3±2.5 mV, n=8, p〈0.24) on the I_Kv activation. Equimolar replacement of EGTA with EDTA, a more potent chelator of Mg²⁺, in the pipette solution also attenuated the shift in Na⁺-free PSS (ΔV_a=-13.7±2, n=12, p〈0.05). However, the addition of the Ca²⁺ chelator BAPTA (10 mM) to the pipette solution did not affect the Na⁺-dependent shift in I_Kv activation (ΔV_a=-18±1.9 mV, n=5, p〉0.3). Our data suggests that Kv channels in PAMs can be potently regulated by Na⁺ dependent mechanism(s) in the physiological range of membrane potentials. Our results are consistent with the inhibition of the Na⁺-Mg²⁺ extrusion mechanism by the removal of extracellular sodium, however, the involvement of other Na⁺-dependent mechanisms such as Na⁺-H⁺ and Na⁺-Ca²⁺ exchangers cannot yet be excluded.