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Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase.
Müllner C
,
Vorobiov D
,
Bera AK
,
Uezono Y
,
Yakubovich D
,
Frohnwieser-Steinecker B
,
Dascal N
,
Schreibmayer W
.
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To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.
Figure 1. Effect of β-adrenergic stimulation on IKACh of rat atrial cells. (A) Original whole cell current recordings at a membrane holding potential of −80 mV. IKACh was induced by superfusion with ACh (10 μmol/liter) or ACh plus Iso (1 μmol/liter) in the absence (top) and in the presence (bottom) of 50 μmol/liter H89. (B) Statistics of the effect of Iso on IKACh amplitude, shown as percent increase compared with IKACh induced by ACh alone. Calculated average values ± SEM are shown; the number of individual cells is given in parenthesis. (* and **) Mean value deviates significantly (P < 0.05 and 0.01) from the H89 treated group. (Left to right) Effect of Iso coapplied with ACh (control, H89 treatment); additional IKACh induced by Iso application during ACh (control, IKACh recorded with nystatin perforated patch, H89 treatment). (C) Effect of Iso coapplication on kinetics of I KACh activation. (Left to right) Regular IKACh induced by ACh superfusion; IKACh induced by ACh coapplied with Iso; same as previous, but in the presence of 50 μmol/liter H89. (Dotted line) Monoexponential fit through the rising phase of I KACh. (Broken line) Biexponential fit, comprising a fast and an additional slow time constant.
Figure 4. Effect of heterologous Gαs overexpression on basal and agonist-induced GIRK currents. (A) Statistics of effect of Gαs overexpression on basal current, normalized to basal IHK current of control (no Gαs overexpressed). Calculated average values ± SEM from two different batches of oocytes are shown (three to five oocytes per batch). Individual experiments were performed either in the absence (white bars) or after preincubation (gray bars) of oocytes in 50 μmol/liter H89. (# and ##) Mean value deviates significantly (P < 0.05 and 0.01) from the corresponding basal current (same amount of Gαs cRNA injected), but without H89 preincubation. (*, **, and ***) Mean value deviates significantly (P < 0.05, 0.01, and 0.001) from the basal current without Gα s overexpression. (B) Same as in A, but agonist-induced GIRK currents are normalized to IACh of control (no Gαs overexpressed).
Figure 2. Stimulation of PKA by β2AR in Xenopus oocytes. (A) Original current trace from an oocyte expressing CFTR Cl− channels and β2AR recorded at a holding potential of −80 mV in ND96 solution. Upon isoproterenol superfusion (1 μmol/liter), CFTR channels were stimulated by PKA phosphorylation, what could be reversed by washing out of the agonist. During the second application of Iso, 30 pmol Rp-cAMPS were injected into the cytosol, abolishing and reversing the effect of agonist. (B) Same as in A, but instead of the second Iso pulse, 30 pmol cAMP was injected, stimulating CFTR channels to the same extend as the Iso pulse. (C) Statistics of stimulation of CFTR by agonist and/or cAMP and analogues normalized to a first, control pulse of Iso (taken as 100%). Calculated average values ± SEM are shown; the number of individual oocytes is given in parenthesis. (***) Mean value deviates significantly (P < 0.001) level from the group injected with stimulatory cAMP analogues. (Iso) After washing out of the first Iso pulse, 5 nl H2O were injected into the oocyte, and then the magnitude of CFTR currents elicited by a second pulse of Iso was measured. (cAMP) After the first pulse of Iso superfusion, 30–60 pmol cAMP (n = 3) or Sp-cAMPS (n = 2) were injected into the cytosol and the resulting CFTR currents were measured. (Rp-cAMPS) Same as cAMP, but Rp-cAMPS was injected instead of stimulatory cyclic nucleotides.
Figure 3. β2-adrenergic facilitation of heterologously coexpressed GIRK currents and cAMP effects in Xenopus laevis oocytes. (A) Original current trace recorded at a membrane holding potential of −80 mV. ND and HK denote changes of the superfusion medium from ND96 to HK, bars denote superfusion with agonists. (B) Same as in A, but Iso was applied alone or during ACh. (C) Same as in A, but 30 pmol cAMP was injected during HK before ACh superfusion. (D) Statistics of the effects of isoproterenol superfusion and/or cytosolic injection of cAMP and Rp-cAMPS on basal current amplitude, expressed as percentage of basal I HK. Calculated average values ± SEM are shown; the number of individual cells is given in parenthesis. (** and ***) Mean value deviates significantly (P < 0.01 and 0.001) from the corresponding control group (i.e., oocytes not expressing heterologous β2AR in the case of Iso effects) or cAMP injection into native oocytes in the case of cAMP effects. (Left to right) Iso-induced current on basal HK (with or without coexpressed β2AR), 30–60 pmol cAMP injection during basal HK, cAMP injection into native oocytes, injection of 30–60 pmol Rp-cAMPS during basal HK. (E) Same as in C, but cAMP was injected during ACh. (F) Current–voltage relation of the cAMP-induced currents as measured by a triangular voltage pulse (f = 1 Hz). (○) Current induced by cytosolic cAMP injection on basal current, obtained by subtraction of the basal current before cAMP injection from basal current after cAMP injection. (•) Current induced by cAMP injections on ACh-induced currents. (G) Statistics of the effects of isoproterenol superfusion and/or cytosolic injection of cAMP on ACh-induced current amplitude, expressed as a percentage of basal IHK. Calculated average values ± SEM are shown; the number of individual cells is given in parenthesis. (** and ***) Mean value deviates significantly ( P < 0.01 and 0.001) from the corresponding control group (i.e., oocytes not expressing heterologous β2AR in the case of Iso effects) or cAMP injection into native oocytes in the case of cAMP effects. (Left to right) Iso effect during ACh (with or without β 2AR), Iso effect upon coapplication with ACh (with or without β2AR), cAMP injection during ACh.
Figure 5. β2-adrenergic facilitation of GIRK currents is not produced by coupling to inhibitory G-proteins. (A) Original current traces recorded at −80 mV. GIRK currents were induced by superfusion of the oocytes with 1 μmol/liter isoproterenol. (Top) Current trace from an oocyte expressing β2ARwt and treated with PTX. (Bottom) Same as in the top, but the oocyte expressed β2AR PF instead of β2ARwt. (B) Statistics of the effect of PTX treatment and/or β2AR mutation on GIRK facilitation, expressed as a percentage of basal IHK of the control (no PTX treatment, β2ARwt expressed). (Empty bars) Oocytes expressing β2ARwt; (hatched bars) oocytes expressing β2ARPF. (+ and −) Oocytes incubated or not treated with PTX. Calculated average values ± SEM from three different batches of oocytes (three oocytes/batch) are shown. (* and ***) Mean value deviates significantly ( P < 0.05 and 0.001) from the corresponding control group; i.e., oocytes without PTX treatment.
Figure 6. cAMP effects in the absence of heterologously coexpressed seven-helix receptors. Original current trace recorded at −80 mV derived from an oocyte expressing exclusively GIRK1/GIRK4 proteins. During HK superfusion, 50 pmol cAMP was injected.
Figure 7. cAMP effects on GIRK isoforms. (A) Original current trace recorded at −80 mV from an oocyte expressing m2R and GIRK1. GIRK currents are therefore produced exclusively from the GIRK1/GIRK5 heterooligomeric assembly. cAMP was injected during basal HK current. (B) Current trace recorded from an oocyte expressing GIRK1/GIRK2 channels (endogenous GIRK5 was knocked out with the KHA2 antisense oligonucleotide). cAMP was injected during ACh superfusion. (C) Same as in B, but the oocyte expressed homooligomeric GIRK2 channels. (D) Effect of antisense oligonucleotide (KHA2) coinjected with either GIRK1 or GIRK1/GIRK4 cRNA on IACh currents. Calculated average values ± SEM are shown; the number of individual cells is given in parenthesis. (* and ***) Mean value deviates significantly (P < 0.05 and 0.001) from the corresponding control group (KHA2 not coinjected). (E) Statistics of cAMP effects on various GIRK subunit combinations on basal IHK currents as well as on agonist-induced currents (IACh). Calculated average values ± standard error of the effect normalized to basal IHK produced by the GIRK1/GIRK4 combination of a given experimental day are shown; the number of individual oocytes are in parenthesis. (** and ***) Mean value of the experimental group deviates significantly (P < 0.01 and 0.001) from zero.
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