Experiments to Test the Role of Phosphatidylinositol 4,5-Bisphosphate in Neurotransmitter-Induced M-Channel Closure in Bullfrog Sympathetic Neurons

Abstract

Various neurotransmitters excite neurons by suppressing a ubiquitous, voltage-dependent, noninactivating K+ conductance called the M-conductance (gM). In bullfrog sympathetic ganglion neurons the suppression of gM by the P2Y agonist ATP involves phospholipase C (PLC). The present results are consistent with the involvement of the lipid and inositol phosphate cycles in the effects of both P2Y and muscarinic cholinergic agonists on gM. Impairment of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) with the phosphatidylinositol 4-kinase inhibitor wortmannin (10 microm) slowed or blocked the recovery of agonist-induced gM suppression. This effect could not be attributed to an action of wortmannin on myosin light chain kinase or on phosphatidylinositol 3-kinase. Inhibition of PIP2 synthesis at an earlier point in the lipid cycle by the use of R59022 (40 microm) to inhibit diacylglycerol kinase also slowed the rate of recovery of successive ATP responses. This effect required several applications of agonist to deplete levels of various phospholipid intermediates in the lipid cycle. PIP2 antibodies attenuated the suppression of gM by agonists. Intracellular application of 20 microm PIP2 slowed the rundown of KCNQ2/3 currents expressed in COS-1 or tsA-201 cells, and 100 microm PIP2 produced a small potentiation of native M-current bullfrog sympathetic neurons. These are the results that might be expected if agonist-induced activation of PLC and the concomitant depletion of PIP2 contribute to the excitatory action of neurotransmitters that suppress gM.

Does diacylglycerol regulate KCNQ channels?

“…First, inhibiting the formation of phosphatidic acid does not bother suppression or recovery. A similar result with R59022 was obtained in bullfrog [24]. Second, all products of DAG metabolism (such as arachidonic acid) must be produced slowly, as DAG is normally metabolized with a time constant of 80 s. This would mean that these products are not generated significantly during the onset of inhibition (<7 s) and start to accumulate only during the recovery phase for short agonist exposures.…”

“…Our point is that such actions would have to be slow to develop after application of a stimulus for PLC. Ford et al [24] did find that if DAG kinase was inhibited with R59022, repeated PLC activation eventually slowed recovery of M current, presumably reflecting the stoppage of phosphatidylinositol regeneration from phosphatidic acid. The possibility of roles for PKC remains a more difficult question.…”

Regulation of Neuronal M-Channel Gating in an Isoform-Specific Manner: Functional Interplay between Calmodulin and Syntaxin 1A

“…Activation of G-protein-coupled receptors of the Gq/11 family strongly suppresses M-channels (Delmas and Brown, 2005), via depletion of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) upon muscarinic receptor stimulation (Suh and Hille, 2002;Ford et al, 2003;Zhang et al, 2003;Suh et al, 2004;Winks et al, 2005) or via Ca 2ϩ -calmodulin (CaM) action on the channels upon bradykinin B 2 and purinergic P 2 Y receptor activation (Cruzblanca et al, 1998;Bofill-Cardona et al, 2000;Gamper and Shapiro, 2003;Zaika et al, 2007). Recently, syntaxin 1A (Syx), a plasma membrane protein component of the exocytotic SNARE complex, shown to interact physically and functionally with different voltage-gated K ϩ channels (Fili et al, 2001;Leung et al, 2003Leung et al, , 2007Michaelevski et al, 2003Michaelevski et al, , 2007Tsuk et al, 2005;Yamakawa et al, 2007;Regev et al, 2009), was suggested to be another blocker of M-channels (Regev et al, 2009).…”

Differential Neurotrophic Regulation of Sodium and Calcium Channels in an Adult Sympathetic Neuron

“…In bullfrogs, there are three distinct GnRH receptor subtypes, all of which appear capable of coupling to G q (Wang et al 2001). Within the sympathetic ganglia, LHRH activates PLC to increase inositol trisphosphate turnover and intracellular Ca 2ϩ concentration (Pfaffinger et al 1988) and to suppress M-type K ϩ current (Ford et al 2003b(Ford et al , 2004, suggesting that the LHRH/GnRH receptor(s) in this tissue is/are also coupled to G q/11 . It is yet to be determined which of the three identified bullfrog GnRH receptors is/are involved in both this process and the neurotrophic effects described in the present work.…”

“…The present experiments were therefore undertaken to determine 1) whether LHRH-induced increase of I Ca in BFSG proceeds via the MAPK pathway in a similar fashion to NGF, 2) how Gprotein-coupled LHRH receptors signal to the MAPK pathway, 3) the mechanism whereby NGF produces long-term increases in I Na , and 4) whether LHRH has access to this transduction mechanisms so that it can produce long-term increases in I Na in the same way as NGF. Parts of this work were published in preliminary reports (Ford and Smith 2000;Lu et al 2002;Wong et al 2006).…”