Voltage-Gated Sodium Channels in Human Aortic Smooth Muscle Cells

Abstract

Whole-cell, voltage clamp methods were used to study inward currents in human aortic smooth muscle cells in culture. Cells were plated on glass coverslips, cultured in supplemented M-199 media with 5% serum and studied as primary cells and at passages 2–5. Inward currents were measured with a pipette solution containing Cs⁺ and TEA⁺ to block K⁺ currents and with 2.5 m M [Ca²⁺]₀ in the perfusate. Inward currents activated at about –50 mV, peaked at about –15 mV and reversed at about +30 mV. Values of peak inward current averaged 14.7 ± 3.3 pA/pF and cell capacitance averaged 124 ± 10 pF (n = 35). These currents activated rapidly with a time-to-peak current of 2.4 ± 0.3 ms at a test potential of –10 mV from a holding potential of –80 mV. The current also inactivated rapidly with a time course that could be described by two components with time constants of 1.8 ± 0.2 and 17.8 ± 3.5 ms at –10 mV. The currents decreased when extracellular Na⁺ was reduced and were completely inhibited by 50 n M tetrodotoxin (TTX), suggesting that they represented voltage-gated Na⁺ currents (I_Na). Activation curves were characterized with a V_0.5 = –16.6 ± 2.4 mV and a slope factor k = –5.2 ± 0.2 mV while inactivation curves were characterized with a V_0.5 = –60.9 ± 1.7 mV and a slope factor k = 8.8 ± 0.4 mV. Lowering external [Ca²⁺] to zero increased the maximum I_Na, shifted its voltage dependence in the hyperpolarizing direction and increased the rate of I_Na inactivation. Increasing external [Ca²⁺] or [Mg²⁺]decreased I_Na and slowed its rate of inactivation. These studies demonstrate the presence of voltage-gated Na⁺ channels with high TTX sensitivity that are modulated by extracellular divalent cations in human aortic smooth muscle cells maintained in cell culture. Window currents were found in the voltage range of –50 to –20 mV, suggesting that these channels could contribute to the resting membrane potential.