Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells

Abstract

1. The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias. 2. Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n-3 polyunsaturated fatty acids (n-3 PUFA) present in fish oils. 3. We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito. 4. The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8 +/- 0.2 and 5.3 +/- 0.4 ms for activation and 20.0 +/- 1.2 and 96.6+/-6.7 ms for inactivation at +45 mV at 23 degrees C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation= -6.7+/-2.5 mV, V1,2 of steady state inactivation= -51.3+/-0.2 mV at 23 degrees C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37 degrees C) compared to room temperature (23 degrees C). 5. The n-3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 micromol L(-1). Blockade of the transient outward current may be an important mechanism by which n-3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia.

Figure 1

(a) Activation of Kv4.3 current. Cells were held at −105 mV and then stepped to test potentials ranging from −75 to +125 mV (in 20 mV increments) for 490 ms. The lower portion of this Figure illustrates this stimulation protocol, and the upper portion shows the resulting current traces. (b) The normalized conductance was plotted as a function of test potential, and the resulting curve was well fit by a Boltzmann sigmoidal function, with V_1/2 of current activation=−6.9±2.9 and slope factor +26.9±2.9 mV (n=10).

Figure 2

(a) Steady state inactivation of Kv4.3 current. Cells were subjected to a conditioning potential of 400 ms duration ranging from −105 to +25 mV (in 10 mV increments) prior to a 300 ms step to +65 mV. (b) Normalized current as a function of conditioning potential. The resultant curve is well fit by a Boltzmann sigmoidal function, with V_1/2 of steady state inactivation of −51.3±0.2 mV and slope factor of −7.2±0.2 mV (n=11).

Figure 3

(a) Time course of recovery from inactivation of Kv4.3 current. Cells were held at a polarized membrane potential and then a 500 ms depolarization to +45 mV was applied (S1). The cell was then returned to the polarized holding potential for a variable time interval (5–905 ms) before a second 500 ms duration stimulus, also to +45 mV was applied (S2). Currents elicited by the second depolarization show exponential recovery. (b) Time course of recovery from inactivation of Kv4.3 current. Current elicited by S2 is normalized to that elicited by S1 and plotted as a function of the interpulse interval duration. Recovery from inactivation follows a monoexponential course, and is slower at more depolarized holding membrane potentials (n=6).

Figure 4

The time course of recovery from inactivation is accelerated at 37°C compared to room temperature, with the time constant of inactivation at −85 mV decreasing from 276 ms (95% C.I. 245–317 ms) to 94 ms (95% C.I. 77–120 ms); n=8, P<0.001.

Figure 5

(a) Raw traces showing block of Kv4.3 current with DHA. In this example, 3 μmol L⁻¹ DHA results in 65% block. (b) Dose-response curve for DHA inhibition of Kv4.3 current by DHA. The IC₅₀ is 3.6 μmol L⁻¹, and the Hill slope is −0.95±0.31 (n=5).

Figure 6

DHA produces a modest acceleration in current inactivation: raw traces from a single cell of current in response to a depolarization to +45 mV showing acceleration of inactivation with 3 μmol L⁻¹ DHA. The τ_f of inactivation decreased from 20.0±1.2 to 11.2±2.3 ms with 3 μmol L⁻¹ DHA (n=6).

Figure 7

Change in the voltage-dependence of Kv4.3 current steady state inactivation with 3 μmol L⁻¹ DHA. The V_1/2 is shifted to the left, from −48.4±1.0 to −67.1±2.2 mV (n=6, P<0.001).