Inward rectifier K channels were first described in skeletal muscle and egg-cell membranes(1, 2) . They are now found in many cell types and are characterized by the following properties: (i) an activation by hyperpolarization negative to the reversal potential for K ( E), (ii) an activation potential shifting with E, (iii) a blockade by Cs and Ba(3) .

A class of inward rectifier K channels is gated via G-proteins (GIRK). In atrial cells, acetylcholine released by stimulation of the vagal nerve causes the opening of a GIRK channel (I) via the activation of a m2-muscarinic receptor. The induced hyperpolarization results in a slowing of cardiac frequency(4, 5) . GIRK channels also exist in a variety of neuronal cells and the modulation of such channels generates slow synaptic potentials(6, 7) . They are coupled to various neurotransmitter receptors such as the muscarinic cholinergic, µ, , and opioid, -adrenergic, somatostatin, substance P, and GABA receptors(8, 9, 10) .

A GIRK channel, termed GIRK1 (11) or KGA(12) , was cloned from rat heart, and two structural homologs were cloned from mouse brain, mGIRK2 and mGIRK3(13) . Another close structural parent of the GIRK family (rcK) was described initially as an ATP-sensitive K channel(14) , i.e. a channel for which activity is controlled by intracellular ATP(15) . However, it has been shown recently that the functional I channel stimulated by the G-protein subunits is a heteromultimer composed of two GIRK subunits, GIRK1 and CIR, a channel subunit which is nearly identical with rcK(16) .

A mouse analog of rcK/CIR that we have cloned, and designed in this paper as mGIRK4, presents the characteristic features of a G-protein-activated inward rectifier K channel. In contrast to mGIRK1, which is present both in heart and brain, mGIRK4/CIR is specifically localized in the heart, whereas mGIRK2 and mGIRK3 are expressed mainly in the brain. By using different strategies, co-localization of transcripts, immunoprecipitation, and electrophysiology, we present evidences for a heterologous GIRK subunit assembly in the brain. The paper also describes the main electrophysiological properties and the modulation by ATP and Na of the currents expressed by mGIRK2 and the mGIRK1 + mGIRK2 combination in Xenopus oocyte.

EXPERIMENTAL PROCEDURES

Isolation of mGIRK2A and mGIRK4/CIR Clones and mGIRK2/3/2 Chimera Construction

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To construct the chimera mGIRK2/3/2, the mGIRK3 sequence was mutated at positions 151 (the A of the initiation codon taken as base 1) and 1012 to introduce MunI and NheI restriction sites, respectively, without modification of the amino-acid coded sequence. Site-directed mutagenesis was performed using oligonucleotide primers according to the manufacturer's protocol (Promega). The central mGIRK3 sequence between these two sites was exchanged with the corresponding mGIRK2 sequence in which a MunI site was created at position 255. The NheI site is found naturally in the mGIRK2 sequence.

Northern Blot Analysis

In Situ Hybridization

Antibody Preparations, Immunoprecipitations of Transfected GIRK Channels

TSA201 cells were transfected with mGIRK2 and mGIRK4/CIR subcloned into the expression vector pcDNA (Invitrogen) by the calcium phosphate method. After 48 h, cells were harvested and microsomes were prepared. Briefly, cells were homogenized in 150 mM NaCl, 3 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.7 µg/ml pepstatin A, and 10 mM Tris-HCl (pH 8) buffer, centrifuged at 1000 g, and the supernatant was pelleted at 100,000 g for 30 min. Pellets were dissolved in the homogenization buffer and stored at -20 °C. Aliquots of 50 µg of microsomes were solubilized in 100 µl of homogenization buffer containing 1% Triton X-100. After 1 h at 4 °C, the volume was adjusted to 400 µl with homogenization buffer (final Triton X-100 concentration of 0.25%). Preimmune or immune sera were added for 3 h at a 200-fold dilution, followed by addition of 10 µl of protein A immobilized on Sepharose CL-4B (Sigma) for 1 h at 4 °C under slow rocking. Pellets were washed five times with the homogenization buffer containing 0.25% Triton. Immunoprecipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide) and transferred onto nitrocellulose membrane (Hybond-C extra, Amersham). Blots were saturated with phosphate-buffered saline containing 3% low-fat dry milk and incubated with affinity-purified guinea pig polyclonal antibodies diluted 400-fold. Blots were revealed with a F(ab`)-purified horseradish peroxidase-conjugated goat anti-guinea pig antibody (Cappel) and then incubated with substrate for ECL (chemiluminescence method, Boehringer).

In Vitro Transcription and Xenopus Oocytes Preparation

Preparation of Xenopus laevis oocytes and cRNA injections have been described previously(20) . 33 ng of GIRK cRNAs and 833 pg of subunit cRNAs were injected per oocyte.

Electrophysiology

RESULTS

Cloning of a Splice Variant of mGIRK2

Figure 1: Sequences and PCR detection of mGIRK2 splice variants. a, nucleotide and deduced amino acid sequences of the carboxyl termini of mGIRK2 splice variants. Nucleotides are numbered from the first initiation ATG codon, and amino acids are numbered beginning with the initiating Met. Nucleotides that differ between mGIRK2 and mGIRK2A are printed in italics. The carboxyl-terminal 11 amino acids specific to the mGIRK2A variant are shown in bold. Sequences corresponding to the oligonucleotides sense (P1 and P2) and antisense (P3 and P4) used in b are boxed. b, reverse transcription-PCR amplification of both splice variants. Specific DNA fragments were amplified from mouse brain cDNA by using P1 and P3 primers for mGIRK2 and P1 and P4 primers for mGIRK2A. PCR products were blotted and probed with the P-labeled P2 oligonucleotide.

The entire coding region of the mGIRK4/CIR cDNA was cloned from heart mouse cDNA (GenBank accession number U33631). It shares 95% and 97.6% sequence identity with the rat CIR (16) /rcKATP (14) at the nucleotide and amino acid levels, respectively. The percentages of amino acid identities between mGIRK4/CIR and the other mGIRKs were 64.3% (mGIRK1), 71% (mGIRK2A), and 70% (mGIRK3). These values fall down to 44% and 46% in comparison between mGIRK4/CIR and ROMK1 and IRK1, respectively. The highest degree of sequence conservation between all these channels was found in a central core, starting approximately 50 residues upstream of the first transmembrane domain and ending 175 residues downstream of the second transmembrane domain.

Distribution of the mGIRK mRNAs

Figure 2: Distribution of GIRK transcripts. a, the expression of GIRK transcripts was analyzed by Northern blot in adult mouse brain (B) and heart (H). Poly(A) RNAs (5 µg/lane) were isolated, blotted, and hybridized as described under ``Experimental Procedures.'' For GIRK2, the DNA probe corresponds to the 5`-coding sequence conserved in the two splice variants. b, dark field photomicrographs of emulsion autoradiograms illustrating the co-expression of mGIRK1 and mGIRK2 transcripts in CA3 pyramidal cells (P) of the hippocampus. Scale bar: 500 µm.

In situ hybridization studies have shown that the gene expression patterns of GIRK1, GIRK2, and GIRK3 are widely distributed in the brain and are very similar ((21, 22) and results not shown). The highest expression levels appeared in the neo- and allocortical regions, hippocampus, olfactory bulb, and cerebellum. The mGIRK4/CIR expression was very low in the adult rat brain (not shown). To determine the potential significance of heteromultimeric formation in the brain, a high resolution study obtained by microscopic analysis of emulsion-dipped sections has been performed. Fig. 2b shows an example of the high degree of overlap of the expression patterns of mGIRK1 and mGIRK2 transcripts. More than 80% of the neurons are labeled with the mGIRK1 and mGIRK2 probes in the CA3 pyramidal cell layer of the hippocampus (Fig. 2b). Coexpression of both transcripts in the same neuron type was also observed in most of the other strongly labeled central nervous system areas and was particularly evident in the granule cells of the dentate gyrus, in granular layers of the cerebellum, and in the mitral cells in the olfactory bulb (not shown). No area expressing only one GIRK mRNA could be clearly detected in the whole brain.

Properties of Macroscopic mGIRK Currents Expressed in Xenopus Oocytes

Similarly to I(23) , expressed mGIRK channels are stimulated by the G-protein dimer. Although the requirement for these G-protein subunits was not systematically observed for heteromultimeric channels (not shown), were always co-injected in the following part of the work. Injections of mGIRK2 or mGIRK1 + mGIRK2 (mGIRK1,-2) into Xenopus oocytes resulted in the expression of inwardly rectifying currents. The expression of mGIRK2 in the absence of other mGIRK subunits was successful only in 35% of the oocyte batches tested. This low expression frequency was nevertheless sufficient to allow a detailed characterization of the biophysical properties of the current. The GIRK current expression frequency reached 100% with combined injections of mGIRK1 and mGIRK2. Fig. 3, a and b, shows superimposed current traces evoked by voltage steps ranging from -135 to +45 mV in 30-mV increments from a holding potential of 0 mV (K equilibrium potential). mGIRK2 (Fig. 3a) and mGIRK1,-2 (Fig. 3b) currents in response to hyperpolarizing voltage steps display different kinetics. mGIRK2 currents activate rapidly, in less than 5 ms, and then partially inactivate with a time constant of 243 ± 15 ms (n = 8) at -130 mV. Activation/inactivation kinetics of mGIRK1,-2 currents were very different, with a slower time constant (81 ± 5 ms at -130 mV, n = 14) and no inactivation.

Figure 3: Whole cell properties of mGIRK2 and mGIRK1,-2 currents. a and b, currents recorded under the double microelectrode technique in oocytes injected with cRNAs coding for mGIRK2 (a) or mGIRK1+ mGIRK2 (mGIRK1,-2) (b), and for the G-protein subunits. The 6-s voltage pulses ranged from -135 mV to +45 mV in 30 mV steps. c, mGIRK2 peak currents recorded in 2, 14, 26, 50, 74, or 98 mM external K. d, relationship between the reversal potential of GIRK2 currents and the external K concentrations. e, currents recorded during voltage steps to -120 mV. f, steady state current-voltage relationship, both in various external Cs concentrations. g, currents recorded during voltage steps to -120 mV. h, steady-state current-voltage relationship, both in various external Ba concentrations. In e, f, g, and h, the cation external concentrations were 1 µM (), 3 µM (), 10 µM (), 30 µM (), 100 µM (), 300 µM (), 1 mM (), and 3 mM ().

As expected for a K-selective inward rectifier (24) , the activation potential of mGIRK2 became more negative as external K concentration decreased and the amount of shift (52.6 ± 0.8 mV, n = 6, for a 10-fold change in external [K] was close to the K equilibrium value (59 mV) estimated from the Nernst equation (Fig. 3, c and d). The shifts in the threshold of activation for mGIRK1,-2 and mGIRK2 + mGIRK4/CIR (mGIRK2,-4) were, respectively, 50.9 ± 2.3 mV (n = 4) and 50.6 ± 3.3 mV (n = 3) for a 10-fold change in external [K] (not shown), consistent with a predominant K selectivity for these channels.

As for the majority of K selective channels, external application of Ba or Cs blocked mGIRK2 currents in a concentration-dependent manner (Fig. 3, e-h). The Cs block was voltage-dependent giving rise to typical bell-shaped I/V curves for potential values negative to -50 mV (Fig. 3f). The mechanism of Ba block, for concentrations less than 1 mM, is probably of the ``open channel block'' type (24) as suggested by the pronounced fast inactivation component of the resulting current (Fig. 3g). The IC values for the Cs inhibition were 94.2 ± 16 µM (n = 3), 94.5 ± 7.6 µM (n = 5), and 94.3 ± 1.3 µM (n = 3) for mGIRK2, mGIRK1,-2, and mGIRK2,-4, respectively, while the IC values for the Ba inhibition were 94.2 ± 3.8 µM (n = 3), 105.7 ± 6.9 µM (n = 6), and 97.9 ± 1.9 µM (n = 3) for mGIRK2, mGIRK1,-2, and mGIRK2,-4, respectively. Other K channels blockers, including tetraethylammonium (3 mM), 4-aminopyridine (100 µM), clofilium (33 µM), tedisamil (50 µM), RP 98886 (30 µM), RP 62719A (30 µM), glibenclamide (10 µM), or K channel openers(15) , such as pinacidil and P1060, both at 100 µM, were without effect on mGIRK2, mGIRK1,-2, and mGIRK2,-4 currents. On the other hand, verapamil and bepridil, two L-type Ca channel blockers, partially inhibited these currents, up to 60% and 40%, respectively, at 100 µM (not shown).

Finally, activation of protein kinase C by the phorbol 12-myristate 13-acetate (30 nM), the diacylglycerol analog, OAG (100 µM), or arachidonate (100 µM), and activation of protein kinase A by forskolin or 8-chloro-cAMP (3 and 300 µM) were without effect on GIRK currents (not shown).

Single-channel Analysis and Rectification Properties

Figure 4: Single-channel properties of mGIRK1,-2 (a, b, and c) or GIRK1,-2A channels (d, e, and f). a and d, current traces recorded in the inside-out configuration at -80 mV and +80 mV in the presence () or the absence () of 10 mM Mg. b and e, mean I-V curves (n = 10) in symmetrical 140 mM K. c and f, open time distribution obtained at -80 mV in 10 mM Mg. The histograms were fitted with single-exponential curves with time constants of 0.21 ms for mGIRK1,-2 and 0.16 ms for mGIRK1,-2A. Currents were filtered at 3.5 kHz. g, time course of the effect of 100 µM spermine on NP(N = number of channels, P = open probability), calculated every 1 s from mean outward currents, in the absence of internal Mg, at +80 mV. h, current traces recorded at the indicated points in g). i, bar graph indicating mean NP values as a function of internal spermine concentration, 100% corresponding to mean NPwithout spermine.

Since a highly voltage-dependent block both by intracellular Mg and by the polyamine spermine have been shown to underlie strong inward rectification in cloned inward rectifiers(25, 26, 27) , we tested the internal spermine dependence of the inward rectification of our expressed channels. In the experiment illustrated in Fig. 4, g and h, inside-out patches containing mGIRK1,-2 channels maintained at +80 mV were first perfused with a Mg-free internal solution leading to an immediate removal of the inward rectification. Then, application of 100 µM spermine led to a complete blockade of the outward current which promptly reappeared after spermine removal. The bar graph (Fig. 4i) shows that the spermine block was dose-dependent with an IC of about 10 µM. Essentially the same spermine effects were obtained on oocytes co-expressing mGIRK1 and the splice variant mGIRK2A (not shown).

Aspartic acid, a negatively charged amino acid present in the second transmembrane domain of inward rectifiers which are not regulated by G-proteins such as IRK1 and BIR10 (positions 172 and 158) has been shown to be implicated in their Mg and spermine sensitivities(25, 27, 28, 29) . The corresponding residue is an aspartate in position 173 in mGIRK1 and a neutral asparagine in position 185 in mGIRK2. To evaluate the importance of the charge at this position for the rectification characteristics of the heteropolymeric G-protein-activated channel mGIRK1,-2, we took advantage of the presence of an asparagine (instead of an aspartate) residue in the corresponding position in mGIRK4/CIR (position 180) as in mGIRK2(13) . The mGIRK2,-4 channel has no negative charge in the positions that have been considered as crucial for Mg- and polyamine-induced inward rectification in IRK channels. Similarly to mGIRK1,-2, mGIRK2,-4 presents an inward rectification in the presence of 10 mM Mg (Fig. 5, a and b). In addition, Fig. 5(d and e) shows that the outward current recorded in a Mg-free solution at +80 mV was totally abolished in the presence of 100 µM spermine. In symmetrical 140 mM K, the unitary conductance was 39 ± 5 pS (n = 5), and the time constant of the open-time distribution in steady-state conditions at -80 mV was 0.51 ms (Fig. 5c).

Figure 5: Single-channel properties and immunoprecipitation of GIRK2,-4 channels. a, inside-out patch recording at -80 mV and +80 mV with () or without () 10 mM internal Mg. b, mean I-V curves (n = 5) in symmetrical 140 mM K. c, open time distribution obtained in the presence of 10 mM Mg, at -80 mV. The histogram was fitted with a single-exponential curve with a time constant of 0.51 ms. d, bar graph indicating the effect of 100 µM spermine on the mean NP, 100% corresponding to mean NP in spermine-free solution. e, current traces recorded in the absence (1) and in the presence (2) of 100 µM spermine. f, immunoprecipitation of mGIRK2 and mGIRK4/CIR subunits from TsA201-transfected cells with pcDNA carrying the indicated GIRK coding sequences. Corresponding microsomes were solubilized in nondenaturing conditions and analyzed by immunoblotting directly or after immunoprecipitation with anti-mGIRK4/CIR rabbit antibodies (lane RK4). Antibodies used to reveal the Western blots were prepared from guinea pig (revealing antibodies K2 and K4).

Immunochemical Demonstration that mGIRK Proteins Form Heteromultimers

ATP Prevents the Rundown of mGIRK Channel Activity

Figure 6: Effect of ATP on GIRK1,-2 activity. a, effect of disodium salt ATP solution (ATP/2Na). Current traces were recorded at -80 mV in the inside-out configuration. b-e, time course of the open probability (NP) calculated every 10 s. b, effect of 10 mM ATP. c, effect on the rundown of the protein kinase A catalytic subunit (40 units/ml). A protein kinase A subunit stock solution was prepared before each experiment at a concentration of 4000 units/ml in the presence of dithiothreitol (6 mg/ml). d, effect of alkaline phosphatase (100 units/ml) on the reactivation of GIRK1,-2. A stock solution was prepared at 2000 units/ml in water. e, effect of the nonhydrolyzable ATP analogue AMP-PCP (10 mM). f, current traces recorded at the indicated numbers in e. Channel open time distribution, calculated from a sample of 1 min in duration, in 10 mM ATP, 0 ATP, and 10 mM AMP-PCP.

Surprisingly, channel activities were maximal when disodium ATP (ATP/2Na) was used instead of Mg-ATP. Fig. 7a shows that mGIRK1,-2 channel activity could be partly restored by application of a 20 mM NaCl in an ATP-free internal solution. To reach maximal channel activity, the simultaneous presence of ATP and Na ions was required. Channel activity was only 20% of the maximal activity on application of 10 mM Mg-ATP in Na-free solution (Fig. 7b). Fig. 7c presents mean results from 5 experiments and clearly shows the synergy of action of ATP and Na in the restoration of channel activity. In the presence of ATP, Li could replace Na for the activation of mGIRK1,-2 channels but with less efficacy (Fig. 7d). The sensitivity to internal Na led us to check if the cloned mGIRK channels could be related to K channels activated by internal Na which have been described in cardiac and neuronal cells(30, 31) . In fact, the only similarity is the requirement of a high concentration of Na (>30 mM) to activate these channels. The high unitary conductance (>100 pS), the impossibility to replace Na by Li for activation, and the specific blockade by R56865 which characterizes the Na-sensitive K channel (32) were not found for mGIRK channels (not shown).

Figure 7: Requirement of Na and ATP for the restoration of GIRK1,-2 channel activity in the inside-out configuration (a-c). In this series of experiments, the ATP used was the ATP-Mg salt. GIRK1,-2 activities were recorded at -80 mV and filtered at 5 Hz. In a and b, initially, patches were internally bathed with an ATP- and Na-free solution. a, effect of 20 mM Na in the absence of ATP. b, effects of 10 mM ATP and 10 mM ATP + 20 mM Na. After the Na removal, note the instantaneous reduction of activity to the level reached in the presence of ATP alone. c, bar graph (n = 5) indicating the respective increase of GIRK1,-2 activities in the presence of Na, ATP, and ATP + Na (taken arbitrary as 100% in each experiment). d, effects of 20 mM Li followed by 20 mM Na in the presence of 10 mM ATP.

Expression of a mGIRK3-mGIRK2 Chimera

Figure 8: Expression of a mGIRK2/mGIRK3 chimeric construct. a, scheme showing the contribution of mGIRK2 sequences (black) in the chimeric construct mGIRK2/3/2. b, bar graph showing the averaged currents recorded at -130 mV in oocytes injected with mGIRK1 and the G-protein subunits together with mGIRK2, mGIRK3, or the mGIRK2/3/2 chimeric assembly (respectively, n = 32, 10, and 12, in 3 to 5 different batches of oocytes). The vertical bars indicate the S.E.

DISCUSSION

Four proteins with structures corresponding to G-protein-gated inward rectifier (11, 12, 13, 14, 16) have been cloned to date. They are designated as mGIRK1, mGIRK2, mGIRK3, and mGIRK4/CIR. mGIRK4/CIR seems to be specific to the heart, and it is not detected in the brain. Conversely, mGIRK2 and mGIRK3 transcripts are specifically present in the brain. mGIRK1 is present at similar levels in heart and brain. In situ hybridization experiments have shown that the distribution of the three mGIRKs is very similar if not identical. Moreover, the colocalization of distinct GIRK transcripts in the same neuronal cells is in agreement with the hypothesis of heteromeric formation. This hypothesis is strongly supported by the tremendous increase of functional expression of GIRK channels when they are co-injected in the same oocyte as compared to single injections.

K channels expressed after the injection of the mGIRK2 cRNA alone or in combination with mGIRK1 cRNA (mGIRK1,-2) or with mGIRK4/CIR cRNA (mGIRK2,-4) in the presence of had the hallmarks of inward rectifier channels: (i) an activation by hyperpolarization negative to the reversal potential for K (E), (ii) an activation potential shifting with E, and (iii) a blockade by Cs and Ba. However, in voltage-clamp conditions, there were some differences between expressions of mGIRK2 and mGIRK1,-2. mGIRK2 currents displayed a rapid activation (<5 ms) and partial inactivation, whereas mGIRK1,-2 channels had a slow activation and did not inactivate. Single-channel analysis of mGIRK2, mGIRK1,-2, and mGIRK2,-4 currents clearly demonstrated that there was only one population of channels with very similar properties characterized by a unitary conductance of about 40 pS and a flickering activity with a mean open time duration of less than 1 ms. The single-channel parameters of mGIRK2 and mGIRK1,-2 were very similar although their activation kinetics at the whole oocyte level were distinct.

A splice variant of mGIRK2 (mGIRK2A) has also been cloned. It has the same sequence as mGIRK2 but contains 11 additional amino acids in the carboxyl-terminal end. mGIRK2A transcripts are also specifically located in the brain. Electrophysiological results have not shown any significant difference between the two forms. Therefore, it is not easy to suggest any specific new function for mGIRK2A. A first possibility would be that the mGIRK2A subunit could associate with other mGIRKs which are not yet discovered. Another possibility is that the different carboxyl-terminal sequences could serve to impose different cellular localizations. Interestingly, the mGIRK2A terminal sequence SKV is very similar to the microbody targeting signal motif SKL(33) .

How do expressed neuronal GIRK channels compare with ``native'' channels? Native GIRK channels recorded in different neuronal cell types have unitary conductances varying from 38 to 55 pS and a time constant of their open-time distribution which is of the order of 2 ms (7, 34) . It then appears that their conductances are similar, but flickering is more rapid for the cloned channels expressed in Xenopus oocytes. However, it should be noted that detailed literature describing neuronal GIRK channel properties at the single-channel level is not yet available. One possibility is that flickering GIRK channels are difficult to record in neuronal membranes where numerous other K channel activities might coexist. Another likely possibility is that some subunit which normally slows down the gating kinetics in native channels is still missing in cloned heteropolymeric channels.

It has been shown previously that the functional cardiac G-protein-activated inward rectifier is in fact composed of an assembly of rat GIRK1 and GIRK4/CIR(16) . The K current expression described above suggests that mGIRK2 can also form heteromultimeric assemblies with mGIRK1 and mGIRK4/CIR. This was actually directly demonstrated by immunoprecipitation studies in the case of the mGIRK2,-4 complex. Experiments using coexpression of mGIRK1 with chimeras of mGIRK3 (which do not express alone or co-injected with mGIRK1, mGIRK2, or mGIRK4/CIR) with the amino- and carboxyl-terminal sequences of mGIRK2 also tend to lead to the same conclusion. The apparent co-localization of mGIRK1 and mGIRK2 in the brain, particularly in CA3 pyramidal cells, is a strong indication that the mGIRK1,-2 complex is a major neuronal GIRK channel. The case of mGIRK3 is not clear. Its lack of expression suggests that it might need a partner that still has to be discovered. One possible partner is the sulfonylurea receptor(35) . ATP-sensitive K channels are present in the brain(36, 37, 38) . They have inward-rectifying properties(23) , are regulated by G-proteins(39, 40) , and may be constituted by the assembly of the protein that binds antidiabetic sulfonylureas (35) and an inward rectifier-type K channel.

After this work was submitted, it was published that the GIRK3 subunit can assemble with GIRK1 and with GIRK2 to either increase (GIRK1) or decrease (GIRK2) their activities(41) . These effects were never seen in our own experiments. These apparently conflicting observations might be explained by assuming that a third, not yet identified, subunit is endogenously present in oocytes and confers the expression properties observed by Kofuji et al.(41) . This component would not be present in our oocytes.

The inward rectification in cloned inward rectifiers (25, 26, 27) is due to a highly voltage-dependent block by intracellular Mg and by polyamines. Mutagenesis experiments have strongly suggested that aspartic acid in position 172 in the inward rectifier IRK1 is pivotal for the effects of Mg and spermine on the inward rectification (25, 26, 27) . This Asp residue is present at corresponding positions in sequences of a number of cloned inward rectifier such as IRK1, mGIRK1, and BIR10(29) , but this residue is replaced by an asparagine in mGIRK2, mGIRK4/CIR, and also in ROMK1(42) . Although they lack this Asp residue, both mGIRK2 and mGIRK4/CIR, when they are expressed independently or when they co-expressed, possess all the hallmarks of inward rectifiers, contrary to ROMK1 which also has an Asn in the corresponding position 171 and which presents a quasilinear I-V relationship. The fact that replacement of Asn-171 by Asp in ROMK1 results in the appearance of a Mg-dependent inward rectification (43) would tend to confirm the important role of an Asp for Mg-dependent inward rectification. However, the fact that the expression of GIRK2,-4, with Asn in the sequences instead of Asp, also leads to a Mg-dependent inward rectifier K channel pleads for the importance of other residues and questions the unique role of this Asp for inducing this inward rectification.

One particularly interesting observation is the requirement of a high concentration of internal ATP (10 mM) in excised patches to prevent a fast rundown of both mGIRK1,-2 and mGIRK2,-4 activities. This ATP dependence would immediately suggest an important role of phosphorylation. However, results presented in this paper show that a kinase activity involving ATP hydrolysis is not implicated as it is for IRK1 and ROMK1 channels(44, 45) . Treatments capable of activating or inhibiting protein kinase A or protein kinase C activity were without effect on the rundown and/or the reactivating action of ATP. Moreover, alkaline phosphatase which would produce a dephosphorylation did not modify the response to ATP. Finally, the activating effects of the nonhydrolyzable ATP analog AMP-PCP on channel activity were similar to if not identical with those of ATP. All these results taken together show that mGIRK1,-2 and mGIRK2,-4 channels are ATP-regulated channels. They require ATP binding to be functional, but ATP hydrolysis is not necessary. ATP binding might occur at the nucleotide-binding site represented by the consensus Walker type A sequence G(X)GK(X)(V/I). This exact motif is missing in mGIRK sequences, but two motives that share similarities with the Walker A consensus sequence are present in the carboxyl-terminal extremities of mGIRK1, mGIRK2, and mGIRK3 subunits. The I(X)GK(X)V motif is present in mGIRK1 and mGIRK2, the V(X)GR(X)V sequence is present in mGIRK3. It has been suggested that similar motives could be implicated in ATP binding(44) . The mGIRK4/CIR sequence does not possess such an ATP consensus sequence.

This paper also shows that internal Na is a regulator of the neuronal mGIRK1,-2 channel activity. This type of property has in fact been observed before with the inward rectifier K channel which is present in starfish eggs (46) . ATP and Na are synergistic in their activating effects. The ATP and Na dependences of neuronal mGIRK activities might be important in neurological diseases. In ischemic situations, or in epileptic seizures, the intracellular ATP concentration drops rapidly while the internal Na concentration increases massively. It is then possible that the function of neuronal GIRK channels will be affected drastically, leading to changes of membrane polarization that might be an important component in a cascade of events leading to very deleterious effects.

FOOTNOTES

*

This work was supported by CNRS, the Association Française contre les Myopathies (AFM), Commission of the European Communities Contract CHRX-CT93-0167, and Bristol-Myers Squibb company for ``Unrestricted Award.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33631[GenBank].

§

To whom correspondence should be addressed: Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. Tel.: 33-93-95-77-00 (or 02); Fax: 33-93-95-77-04; douy@unice.fr.

()

The abbreviations used are: PCR, polymerase chain reaction; GST, glutathione S-transferase; AMP-PCP, adenosine 5`-(,-methylene)triphosphate.

ACKNOWLEDGEMENTS

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