Activation of a PTX-insensitive G protein is involved in histamine-induced recombinant M-channel modulation

Abstract

The M-type potassium current (I(M)) plays a dominant role in regulating membrane excitability and is modulated by many neurotransmitters. However, except in the case of bradykinin, the signal transduction pathways involved in M-channel modulation have not been fully elucidated. The channels underlying I(M) are produced by the coassembly of KCNQ2 and KCNQ3 channel subunits and can be expressed in heterologous systems where they can be modulated by several neurotransmitter receptors including histamine H(1) receptors. In HEK293T cells, histamine acting via transiently expressed H(1)R produced a strong inhibition of recombinant M-channels but had no overt effects on the voltage dependence or voltage range of I(M) activation. In addition, the modulation of I(M) by histamine was not voltage sensitive, whereas channel gating, particularly deactivation, was accelerated by histamine. Non-hydrolysable guanine nucleotide analogues (GDP-beta-S and GTP-gamma-S) and pertussis toxin (PTX) treatment demonstrated the involvement of a PTX-insensitive G protein in the signal transduction pathway mediating histamine-induced I(M) modulation. Abrogation of the histamine-induced modulation of I(M) by expression of a C-terminal construct of phospholipase C (PLC-beta1-ct), which buffers activated Galpha(q/11) subunits, implicates this G protein alpha subunit in the modulatory pathway. On the other hand, abrogation of the histamine-induced modulation of I(M) by expression of two constructs which buffer free betagamma subunits, transducin (Galphat) and a C-terminal construct of a G protein receptor kinase (MAS-GRK2-ct), implicates betagamma dimers in the modulatory pathway. These findings demonstrate that histamine modulates recombinant M-channels in HEK293T cells via a PTX-insensitive G protein, probably Galpha(q/11), in a similar manner to a number of other G protein-coupled receptors. However, histamine-induced I(M) modulation in HEK293T cells is novel in that betagamma subunits in addition to Galpha(q/11) subunits appear to be involved in the modulation of KCNQ2/3 channel currents.

Figure 1. Inhibition of KCNQ2/3 channel currents by histamine via histamine H₁ receptors

A, superimposed KCNQ2/3 channel currents elicited from a HEK293T cell cotransfected with KCNQ2/3 and H₁R cDNAs in the absence (○) and presence (•) of 10 μm histamine, recorded using the nystatin perforated-patch technique. The dashed line represents the zero current level. The cell was held at -20 mV and then hyperpolarized to -50 mV to elicit typical slow inward current relaxation. B, time course of histamine effect in the same cell showing the reversible inhibition of I_M.

Figure 2. Voltage-independent inhibition of histamine

A, families of currents elicited by depolarizing steps from -70 mV to +40 mV from a holding potential of -60 mV in the absence and presence of 1 μm histamine, recorded using the nystatin perforated-patch technique. B, tail currents shown on a faster time scale. C, normalized current-voltage relationship of M-channels in the absence (○) and presence (•) of histamine. The peak current at each voltage step was normalized to the peak current elicited by the depolarizing step to +40 mV in the absence of histamine. D, the mean percentage inhibition of tail currents was plotted against each voltage step to demonstrate the voltage-independent inhibition of I_M by histamine. Error bars indicate the s.e.m.

Figure 3. Activation curves in the absence (○) and presence (•) of 1 μm histamine

Curves were obtained from tail currents recorded at -60 mV following voltage steps from -70 mV to +40 mV as shown in Fig. 2B. The activation curves were fitted with a modified Boltzmann equation (see text). Error bars are the s.e.m.

Figure 4. Effects of histamine on I_M activation

A and B, the activation phase of I_M elicited by a 2 s depolarizing step from -60 mV to -20 mV in the absence and presence of 10 μm histamine, respectively. Currents were recorded using the nystatin perforated-patch technique. The currents are plotted at 1/50th of the original density. The continuous lines are fits to a double exponential function (see text). The dashed lines represent the zero current level. The residuals calculated from the difference between the original record and the fitted line are plotted below each record.

Figure 5. Effects of histamine on I_M deactivation

A and B, the deactivation tail current of I_M elicited by repolarizing the membrane to -50 mV after a 2 s depolarizing step to -20 mV from -60 mV in the absence and presence of 10 μm histamine, respectively. Currents were recorded using the nystatin perforated-patch technique. The currents are plotted at 1/50th of the original density. The continuous lines are fits to a double exponential function (see text). The dashed lines represent the zero current level. The residuals calculated from the difference between the original record and the fitted line are plotted below each record. C and D, the effects of histamine on the fast and slow time constants, respectively. The numbers in parentheses represents the number of cells tested, *P < 0.05 compared with control. Error bars are the s.e.m.

Figure 6. Effects of intracellular GTP and GDP analogues on the histamine-induced inhibition of KCNQ2/3 currents recorded using the open-tip configuration

A-C, KCNQ2/3 currents before (○), during (•) and after (□) 10 μm histamine application with 0.5 mm GTP (A), 0.5 mm GTPγS(B) or 2 mm GDPβS (C) added to the intracellular solution. The current traces were elicited by a 1 s step to -50 mV from a holding potential of -20 mV. The dashed lines represent the zero current level. The lower panel shows the time course of the histamine effect on M-currents elicited from the corresponding cells.

Figure 7. PTX does not prevent KCNQ2/3 channel inhibition by histamine

A, I_M channel currents in the absence (○) and presence (•) of 10 μm histamine elicited from a HEK293T cell pretreated with PTX, recorded using the nystatin perforated-patch technique. The current traces were elicited by a 1 s step to -50 mV from a holding potential of -20 mV. The dashed line represents the zero current level. B, mean inhibition of I_M by 10 μm histamine with and without PTX treatment. The numbers in parentheses represent the number of cells tested. Error bars are the s.e.m.

Figure 8. Expression of PLC-β1-ct reduces the histamine modulation of KCNQ2/3 channels in HEK 293T cells

A and B, I_M in the absence (○) and presence (•) of 10 μm histamine showing the effect of PLC-β1-ct (2.5 ng and 500 ng transfection concentration, respectively) on histamine-induced inhibition. The current traces were elicited by a 1 s step to -50 mV from a holding potential of -20 mV and recorded using the nystatin perforated-patch technique. The dashed lines represent the zero current level. C, concentration-response relationship of PLC-β1-ct effect on histamine-induced I_M inhibition. The numbers in parentheses represent the number of cells tested, *P < 0.05 compared with control. Error bars are the s.e.m.

Figure 9. Expression of RGS2 fails to prevent histamine modulation of KCNQ2/3 channels

A, I_M elicited from a RGS2-expressing HEK293T cell in the absence (○) and presence (•) of histamine. The current traces were elicited by a 1 s step to -50 mV from a holding potential of -20 mV using the nystatin perforated-patch technique. The dashed line represents the zero current level. B, mean inhibition of KCNQ2/3 currents by 10 μm histamine in control and RGS2-expressing cells. The numbers in parentheses represent the number of cells tested. Error bars are the s.e.m.

Figure 10. Expression of Gαt reduces histamine-induced modulation of KCNQ2/3 channels

A and B, I_M in the absence (○) and presence (•) of 10 μm histamine showing the effect of Gαt on histamine-induced inhibition at two different concentrations (2.5 ng and 500 ng transfection concentration, respectively). The current traces were elicited by a 1 s step to -50 mV from a holding potential of -20 mV using the nystatin perforated-patch technique. The dashed lines represent the zero current level. C, concentration-response relationship of Gαt on histamine-induced I_M inhibition. The numbers in parentheses represents the number of cells tested, *P < 0.05 compared with control. Error bars are the s.e.m.

Figure 11. Expression of MAS-GRK2-ct attenuates histamine-induced modulation of KCNQ2/3 channels

A, I_M elicited from a MAS-GRK2-ct -expressing HEK293T cell in the absence (○) and presence (•) of histamine. The current traces were elicited by a 1 s step to -50 mV from a holding potential of -20 mV. The dashed line represents the zero current level. B, mean inhibition of KCNQ2/3 currents by histamine in control and MAS-GRK2-ct-expressing cells. The numbers in parentheses represent the number of cells tested, *P < 0.05 compared with control. Error bars are the s.e.m.