Tetraethylammonium (TEA) increases the inactivation time constant of the transient K+ current in suprachiasmatic nucleus neurons

Abstract

Identifying the mechanisms that drive suprachiasmatic nucleus (SCN) neurons to fire action potentials with a higher frequency during the day than during the night is an important goal of circadian neurobiology. Selective chemical tools with defined mechanisms of action on specific ion channels are required for successful completion of these studies. The transient K(+) current (I(A)) plays an active role in neuronal action potential firing and may contribute to modulating the circadian firing frequency. Tetraethylammonium (TEA) is frequently used to inhibit delayed rectifier K(+) currents (I(DR)) during electrophysiological recordings of I(A). Depolarizing voltage-clamped hamster SCN neurons from a hyperpolarized holding potential activated both I(A) and I(DR). Holding the membrane potential at depolarized values inactivated I(A) and only the I(DR) was activated during a voltage step. The identity of I(A) was confirmed by applying 4-aminopyridine (5 mM), which significantly inhibited I(A). Reducing the TEA concentration from 40 mM to 1 mM significantly decreased the I(A) inactivation time constant (tau(inact)) from 9.8+/-3.0 ms to 4.9+/-1.2 ms. The changes in I(A)tau(inact) were unlikely to be due to a surface charge effect. The I(A) amplitude was not affected by TEA at any concentration or membrane potential. The isosmotic replacement of NaCl with choline chloride had no effect in I(A) kinetics demonstrating that the TEA effects were not due to a reduction of extracellular NaCl. We conclude that TEA modulates, in a concentration dependent manner, tau(inact) but not I(A) amplitude in hamster SCN neurons.

Figure 1

Effects of TEA and 4-AP on I_A of hamster SCN neurons. The bath solution contained TTX (0.5 µM), picrotoxin (50 µM), TEA (40 mM) and a low extracellular Ca²⁺ concentration (0.2 mM) to reduce Ca²⁺ currents. A. Currents were activated by sequence of voltage steps to potentials between −30 mV and +60 mV from a holding potential of −100 mV. This depolarization protocol activated both I_DR and I_A. B. Holding the membrane potential at −40 mV and applying a sequence of voltage steps to depolarized values between −30 mV to +60 mV activated the I_DR. C. Digital subtraction of the currents recorded in A and B. D. Example of the inhibition of I_A by 4-AP (5 mM). The currents shown were recorded each minute during an 8 min 4-AP application. E. A high concentration of TEA (40 mM) did not alter I_A amplitude. Each bar represents the mean I_A amplitude at each step potential (mean ± standard deviation, n = 10). F. a. TEA (40 mM) added to the recording chamber (0 min) produced a progressive decrease in the I_DR activated from a holding potential of −80 mV by a voltage step to +60 mV. Note that the remaining current shows a rapid activation followed by inactivation. I_A recorded during the same experiment. I_A τ_inact increased from 3.3 ms to 28 ms during the 8 min TEA application (36 °C). b. A similar experiment performed at 22°C. TEA (40 mM) increased I_A τ_inact from 1.3 ms to 10.4 ms.

Figure 2

Sensitivity of I_A τ_inact to TEA (40 mM). A, B. Example of the calculated I_A τ_inact. I_A (gray lines) and the black lines the fit to the equation $i(t)=i_0+K{e}^{-\frac{t}{\tau }}$ (see Experimental Procedures). B. The value of the I_A τ_inact was directly related to the value of the membrane potential (Vmem). C. The effect of high (40 mM) and low (1 mM) TEA concentrations on parameters of I_A τ_inact. Distribution of the values for the parameter (a) and I_A τ_inact calculated from the linear fits to the data from each neuron. The parameter (a) represents the voltage dependence of I_A τ_inact while parameter (b) represents the I_A τ_inact at 0 mV. D. The mean ± standard deviation of I_A τ_inact in the presence of either 40 mM or 1 mM TEA shows the strong effect of the TEA on I_A inactivation kinetics.

Figure 3

Determination of I_A τ_inact at holding potentials of −50 mV, −40 mV and −30 mV. A. The gray lines were I_A and the black lines the fit to the equation $i(t)=i_0+K{e}^{-\frac{t}{\tau }}$ B. The calculated I_A τ_inact was not affected by the holding potential. C. The mean ± standard deviation of I_A τ_inact recorded at three holding potentials in the presence of high (40 mM) and low (1 mM) TEA concentration.