Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K ϩ current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using wholecell clamp, we studied tsA-201 cells in which cloned KCNQ2/ KCNQ3 channels were coexpressed with M 1 muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca 2ϩ transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca 2ϩ signals were abolished by the combined use of strong intracellular Ca 2ϩ buffers, an inhibitor of IP 3 receptors, and thapsigargin to deplete Ca 2ϩ stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca 2ϩ as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism. Key words: K ϩ channel; muscarinic receptor; G-protein; calcium; patch clamp; M current A diverse family of neurotransmitters and hormones regulates Ca 2ϩ and K ϩ channels via G-protein-mediated signaling pathways (Wickman and Clapham, 1995;Brown et al., 1997;Dolphin, 1998). Nearly 20 years ago, several investigators gave the name M current to a noninactivating K ϩ current with slow kinetics in sympathetic neurons that is strongly suppressed by muscarinic acetylcholine receptor (mAChR) agonists (Brown and Adams, 1980;Constanti and Brown, 1981). The M current is thought to play an important role in neuronal excitability, and its suppression increases responses to excitatory synaptic inputs (Jones et al., 1995;Wang and McKinnon, 1995). Modulation of the M current by muscarinic receptor agonists and by angiotensin (Constanti and Brown, 1981; Marrion, 1997;Shapiro et al., 1994a) is mediated by a G-protein of the G q /11 class (Delmas et al., 1998; Haley et al., 1998) via a diffusible cytoplasmic second messenger (Selyanko et al., 1992) that is yet to be identified.Although the buffering of intracellular free Ca 2ϩ ([Ca 2ϩ ] i ) to very low levels prevents muscarinic suppression of the M current in rat sym...

Desensitization of cannabinoid receptor signaling by a G-protein coupled receptor kinase (GRK) was examined using the Xenopus oocyte expression system. Application of a CB1 agonist, WIN 55,212-2, evoked a concentration-dependent increase in K ϩ conductance (K ir 3) in oocytes coexpressing rat CB1 with the G-protein-gated, inwardly rectifying K ϩ channels K ir 3.1 and K ir 3.4. Desensitization was slight during continuous agonist application in the absence of GRK and arrestin. However, coexpression of GRK3 and ␤-arrestin 2 (␤-arr2) caused profound homologous CB1 receptor desensitization, supporting the hypothesis that GRK3 and ␤-arr2 effectively produce CB1 receptor desensitization. To identify the regions of the CB1 receptor responsible for GRK3-and ␤-arr2-mediated desensitization, we constructed several CB1 receptor mutants. Truncation of the C-terminal tail of CB1 receptor at residue 418 (⌬418) almost completely abolished desensitization but did not affect agonist activation of K ir 3. In contrast, truncation at residues 439 and 460 did not significantly affect GRK3-and ␤-arr2-dependent desensitization. A deletion mutant (⌬418-439) did not desensitize, indicating that residues within this region are important for GRK3-and ␤-arr2-mediated desensitization. Phosphorylation in this region was likely involved in desensitization, because mutation of either of two putative phosphorylation sites (S426A or S430A) significantly attenuated desensitization. CB1 receptors rapidly internalize after activation by agonist. Phosphorylation of S426 or S430 was not necessary for internalization, because the S426A/S430A CB1 mutant internalized when stably expressed in AtT20 cells. These studies establish that CB1 desensitization can be regulated by a GRK and that different receptor domains are involved in GRK-and ␤-arrestin-dependent desensitization and CB1 internalization.

1 KCNQ K + channels are thought to underlie the M current of neurons. To probe if the KCNQ2 and KCNQ3 subtypes underlie the M current of rat superior cervical ganglia (SCG) neurons and of hippocampus, we raised speci®c antibodies against them and also used the cysteine-alkylating agent N-ethylmaleimide (NEM) as an additional probe of subunit composition. 2 Tested on tsA-201 (tsA) cells transfected with cloned KCNQ1-5 subunits, our antibodies showed high anity and selectivity for the appropriate subtype. The antibodies immunostained SCG neurons and hippocampal sections at levels similar to those for channels expressed in tsA cells, indicating that KCNQ2 and KCNQ3 are present in SCG and hippocampal neurons. Some hippocampal regions contained only KCNQ2 or KCNQ3 subunits, suggesting the presence of M currents produced by channels other than KCNQ2/3 heteromultimers. 3 We found that NEM augmented M currents in SCG neurons and KCNQ2/3 currents in tsA cells via strong voltage-independent and modest voltage-dependent actions. Expression of individual KCNQ subunits in tsA cells revealed voltage-independent augmentation of KCNQ2, but not KCNQ1 nor KCNQ3, currents by NEM indicating that this action on SCG M currents likely localizes to KCNQ2. Much of the voltage-independent action is lost after the C242T mutation in KCNQ2. 4 The correspondence of NEM eects on expressed KCNQ2/3 and SCG M currents, along with the antibody labelling, provide further evidence that KCNQ2 and KCNQ3 subunits strongly contribute to the M current of neurons. The site of NEM action may be important for treatment of diseases caused by under-expression of these channels.

We mutated a conserved aspartate in the second transmembrane domain of the cannabinoid CB(1) receptor to asparagine (D164N), stably transfected it into AtT20 cells, and examined the coupling of this mutant receptor to several intracellular effectors that are targets of wild-type CB(1) receptor activation. We found that the D164N receptor binds the CB(1) agonist WIN 55,212-2 with an affinity matching that of the wild-type CB(1) receptor and inhibits Ca(2+) currents and cAMP production with an equivalent potency and efficacy. This mutation, however, blocks coupling of the receptor to the potentiation of inwardly rectifying potassium channel (KIR) currents and prevents internalization of the receptor after exposure to agonist. Although the mutant receptor did not internalize, we found it was still capable of activating p42/44 MAP kinase. In addition, we made a reciprocal mutation that exchanged the aspartate with an asparagine in the seventh transmembrane region (D164N/N394D). In other seven-membrane-spanning receptors, this reciprocal mutation is known to restore functions disrupted by the mutation of the single conserved aspartate. However, activation of D164N/N394D did not potentiate KIR current, nor did it internalize. We conclude that D164 is necessary for potentiation of KIR current and internalization of receptor but not necessary for agonist binding, inhibition of cAMP production, inhibition of Ca(2+) currents, or activation of p42/44 MAP kinase. Furthermore, CB(1) receptor internalization is not necessary for MAP kinase activation.