Modulation of voltage-gated channel currents by harmaline and harmane

Abstract

Harmala alkaloids are endogenous substances, which are involved in neurodegenerative disorders such as M. Parkinson, but some of them also have neuroprotective effects in the nervous system. While several sites of action at the cellular level (e.g. benzodiazepine receptors, 5-HT and GABA(A) receptors) have been identified, there is no report on how harmala alkaloids interact with voltage-gated membrane channels. The aim of this study was to investigate the effects of harmaline and harmane on voltage-activated calcium- (I(Ca(V))), sodium- (I(Na(V))) and potassium (I(K(V)))-channel currents, using the whole-cell patch-clamp method with cultured dorsal root ganglion neurones of 3-week-old rats. Currents were elicited by voltage steps from the holding potential to different command potentials. Harmaline and harmane reduced I(Ca(V)), I(Na(V)) and I(K(V)) concentration-dependent (10-500 microM) over the voltage range tested. I(Ca(V)) was reduced with an IC(50) of 100.6 microM for harmaline and by a significantly lower concentration of 75.8 microM (P<0.001, t-test) for harmane. The Hill coefficient was close to 1. Threshold concentration was around 10 microM for both substances. The steady state of inhibition of I(Ca(V)) by harmaline or harmane was reached within several minutes. The action was not use-dependent and at least partly reversible. It was mainly due to a reduction in the sustained calcium channel current (I(Ca(L+N))), while the transient voltage-gated calcium channel current (I(Ca(T))) was only partially affected. We conclude that harmaline and harmane are modulators of I(Ca(V)) in vitro. This might be related to their neuroprotective effects.

Figure 1

Raw traces of voltage-activated calcium (a, b), sodium (c, d) and potassium channel currents (e, f) elicited by a depolarisation from the holding potential of −80 to 0 mV (upper trace). Currents under control conditions (black; lower traces) and after blockade of the channel currents (grey) by 100 μM harmaline (a, c, e) or 100 μM harmane (b, d, f) are superimposed.

Figure 2

Dose–response relationship for the block of I_Ca(V) by harmane (dashed line) and harmaline (black line) for a depolarising voltage jump from the holding potential of −80 to 0 mV for 100 ms. IC₅₀'s were 100.6 μM for harmaline and 75.8 μM for harmane. Threshold concentration was below 10 μM. For comparison, data for 100 and 500 μM harmane and harmaline on voltage-activated sodium and potassium channel currents are included (open diamonds are harmaline on potassium channel currents, open triangles are harmane on potassium channel currents, filled diamonds are harmaline on sodium currents and filled triangles are harmaline on sodium currents).

Figure 3

Normalised I–V relation of the effect of harmaline (a, c, e) and harmane (b, d, f) on I_Ca(V) (a, b), I_Na(V) (c, d) and I_K(V) (e, f). Control I–V's are represented by solid lines, effects of 100 μM of either substance are shown by dashed lines and the effects of 500 μM (for I_Na(V) and for I_K(V)) by dotted lines.

Figure 4

Relative reduction of I_Ca(V) (a, b), I_Na(V) (c, d) and I_K(V) (e, f) by harmaline (a, c, e) and harmane (b, d, e) (100 or 500 μM each) over the voltage range tested. Data were calculated from the I–V relations as shown in Figure 3.

Figure 5

Averaged time course with standard deviation of the effect of harmaline (a; n=8) and harmane (b; n=4) on peak I_Ca(V). Currents were elicited by a depolarisation from the holding potential of −80 to 0 mV. The first and second arrows in both panels mark the application of 25 and 100 μM of either substance, respectively. The third arrow indicates the beginning of wash-out of harmaline of harmane.

Figure 6

Absence of a use-dependent reduction of I_Ca(V) by harmaline (A; 25 μM) and harmane (B; 100 μM). The voltage-driven activation of channel was interrupted for several minutes, while substances were applied (first arrow). When the activation was initiated after 350 s, a stable steady state had already been reached. Withdrawal of drugs (second arrow) resulted in a recovery of I_Ca(V).

Figure 7

I–V relation for the sustained calcium channel current (I_Ca(L+N)), taken at the end of the depolarisation (a, b) and for the calculated transient peak current I_Ca(T) (c, d). Control curves (solid lines) and of the relative effect of harmaline (a, c) and harmane (b, d), both 100 μM (broken lines), are shown. Note that sustained and calculated peak voltage-activated calcium channel currents were differentially affected. While the sustained current was maximally reduced at depolarised voltages above −10 mV, the calculated peak current was mainly unaffected over the voltage range tested.