TRPC Channels and Hypothalamic Control of Homeostatic Functions

Abstract

Recent molecular biological and electrophysiological studies have identified multiple transient receptor potential (TRP) channels in hypothalamic neurons as critical modulators of homeostatic functions. In particular, the canonical transient receptor potential channels (TRPC) are expressed in hypothalamic neurons that are vital for the control of fertility and energy homeostasis. Classical neurotransmitters such as serotonin and glutamate and peptide neurotransmitters such as kisspeptin, neurokinin B (NKB) and pituitary adenylyl cyclase-activating polypeptide (PACAP) signal through their cognate G protein-coupled receptors (GPCRs) to activate TPRC 4, 5 channels, which are essentially ligand-gated calcium channels. In addition to neurotransmitters, circulating hormones like insulin and leptin signal through insulin receptor (InsR) and leptin receptor (LRb), respectively, to activate TRPC 5 channels in hypothalamic arcuate nucleus proopiomelanocortin (POMC) and kisspeptin (Kiss1 ^ARH) neurons to have profound physiological (excitatory) effects. Besides its overt depolarizing effects, TRPC channels conduct calcium ions into the cytoplasm, which has a plethora of downstream effects. Moreover, not only the expression of Trpc5 mRNA but also the coupling of receptors to TRPC 5 channel opening are regulated in different physiological states. In particular, the mRNA expression of Trpc5 is highly regulated in kisspeptin neurons by circulating estrogens, which ultimately dictates the firing pattern of kisspeptin neurons. In obesity states, InsRs are “uncoupled” from opening TRPC 5 channels in POMC neurons, rendering them less excitable. Therefore, in this review we will focus on the critical role of TRPC 5 channels in regulating the excitability of Kiss1^ARH and POMC neurons in different physiological and pathological states.

Introduction

There are seven members of the mammalian canonical transient receptor potential (TRPC) channel family. Termed TRPC1–7, they serve as receptor-operated channels, and are similar to the TRP channels involved in Drosophila phototransduction¹. Save for TRPC 2, these channels can be found throughout the mammalian brain². The TRP channels consist of subunits with six membrane-spanning domains that are arranged to form tetrameric complexes similar to that seen with K⁺ channels^3,4. TRPC channels come together as heteromeric channels comprising the TRPC 1, 4 and 5 sub-family^5,6 as well as TRPC 3, 6 and 7 sub-family^7,8. TRPC 4 and 5 share ~73% homology, and TRPC 3, 6 and 7 share ~75% homology¹. TRPC5 channels are essentially ligand-activated calcium channels with a high permeability to calcium (P_Ca/P_Na = 9:1)². TRPC 4 and 5 channels are unique in that they are accentuated by the lanthanide lanthanum (La³⁺), which antagonizes TRPC 3, 6 and 7 channels⁴. This unique feature has been useful in characterizing TPRC 5 signaling in POMC neurons^9,10.

Mammalian TRPC channels can be activated by G protein-coupled receptors and receptor tyrosine kinases^1,11, and they are a major downstream effector of group I metabotropic glutamate receptor (mGluR1) activation in CNS neurons^8,12–14. In nigrostriatal dopamine neurons, mGluR1 agonists induce a current that exhibits the telltale double-rectifying current-voltage plot of TRPC channel activation¹². In the hypothalamus, TRPC 5 channels are the target of not only monoamine (serotonin) and peptide (neurokinin B, pituitary adenylyl cyclase-activating polypeptide) neurotransmitters signaling via classical G protein-coupled receptor mediated pathways, but metabolic hormones like insulin and leptin signaling via InsR and LRb, respectively^9,10,15,16. The peptide receptors are Gq-coupled to phospholipase C (PLC) activation that leads to hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) to inositol 1,4,5 triphosphate (IP₃) and diacylglycerol (DAG), which opens TRPC 5 channels. Recent cryo-EM studies have confirmed that the gating of TRPC 5 is dependent on DAG binding¹⁷.

On the other hand, the metabolic hormones insulin and leptin have more circuitous signaling pathways. Insulin binding to its receptor (InsR) causes phosphorylation of insulin receptor substrate (IRS) proteins that activate phosphatidylinositide 3 kinase (PI3K). Activation of PI3K generates PIP₃, which stimulates phospholipase C (PLC) and protein kinase B (Akt)^10,18–20. PLC then hydrolyzes PIP₂ to DAG, which modulates TRPC 5 channel opening (Figure 1)^10,17,21,22. In POMC neurons InsR couples to specifically to PI3K p110β activation^23,24, and the InsR-mediated excitation of POMC neurons is abrogated by inhibition of PI3K activity^9,10,24,25. In addition, PI3K activity rapidly increases the insertion of TRPC 5 channels into the plasma membrane from intracellular vesicular pools to boost depolarization and Ca²⁺ entry into neurons²⁶. Collectively, all of these PI3K-mediated effects are critically involved in the activation of TRPC 5 channels by insulin in POMC neurons²⁷. Likewise, leptin binding to its receptor (LRb) activates a Janus kinase 2-IRS-PI3K signaling pathway to generate DAG to open TRPC5 channels (Figure 1)⁹.

Figure 1. A cellular model of PACAP-, serotonin-, insulin-, and leptin-signaling leading to TRPC5 channel opening in POMC neurons.

Insulin signals via InsR-IRS-PI3K to phosphorylate PIP₂ and generate PIP₃ that activates phospholipase C (PLCγ) to cause hydrolysis of PIP₂ to inositol 1,4,5 triphosphate (IP₃) and diacylglycerol (DAG), which opens TRPC 5 channels. Similarly, leptin binding to its receptor (LRb) triggers the recruitment of the tyrosine kinase Janus kinase (JAK) 2, leading to phosphorylation of IRS and activation of PI3K, which results in TRPC 5 channel opening via the pathway described above. On the other hand, PACAP and serotonin bind to their cognate Gq-coupled receptors, PAC1R and 5HT2C respectively, to activate phospholipase C (PLCβ), which hydrolyzes PIP₂ to IP₃ and DAG, which opens TRPC 5 channels.

TRPC 5 channel signaling in reproductive circuits

Arcuate Kiss1 (Kiss1^ARH) neurons

Since their discovery in the early part of this century, Kiss1^ARH neurons have been proposed to be the “pulse-generator” neurons that stimulate pulsatile secretion of GnRH^28,29. Fortuitously, the advent of optogenetics at about the same time allowed physiologists to address this hypothesis directly³⁰. Indeed, optogenetic stimulation of Kiss1^ARH neurons expressing channelrhodopsin (ChR2), was shown to generate pulsatile release of LH, presumably through the release of kisspeptin that stimulated the release of GnRH into the median eminence in mice³¹. The hypothalamic cellular circuitry mediating this response was elucidated one year later using a Kiss1^Cre mouse expressing ChR2 (i.e., Kiss1^Cre mice injected with an AAV-DIO-ChR2:mCherry virus)³². Low frequency (1 Hz) photostimulation of the ChR2 fibers evoked a glutamatergic fast EPSC in adjacent but also contralateral Kiss1^ARH neurons, and high frequency stimulation (20 Hz) generated a slow EPSP, which was presumably due to peptide release³³. Indeed, NKB receptor antagonists blocked the optogenetic response³². Moreover, dual patch recording revealed that Kiss1^ARH neurons were excited simultaneously with photostimulation of the contralateral Kiss1^ARH neuronal input³². Thus, focal high frequency autoexcitation of Kiss1^ARH neurons was able to recruit Kiss1^ARH neurons bilaterally to induce simultaneous firing (synchronization) of this critical neural network that is thought to underlie pulse generator activity in mammals^32,34.

Based on the in vitro studies cited above, there was overwhelming evidence that NKB (Tac2) was a critical player in generating the synchronous firing of Kiss1^ARH neurons. In general, tachykinins such as NKB interact with three tachykinin G protein-coupled receptors, Tacr1, Tacr2 and Tacr3. mRNAs for Tacr1 and Tacr3, but not Tacr2, are expressed in Kiss1^ARH neurons based on single cell RT-PCR analysis³⁵, and the depolarizing effect of NKB in Kiss1^ARH neurons is abrogated by TacR3 antagonists^32,36. Since TRPC 5 channels, which have unique biophysical and pharmacological properties (see below), are highly expressed in Kiss1^ARH neurons¹⁶, we hypothesized that TacR3 is coupled to the opening of TRPC 5 channels. Indeed, the TacR3 agonist senktide generates an inward current, which is antagonized by the TRPC channel blocker 2-aminoethoxydiphenyl borate (2-APB)¹⁶. The senktide-induced cation current shows the characteristic double-rectification of the TRPC 5 channel conductance as seen from the I-V plot; with a reversal of −10 mV. Dynorphin is co-expressed in Kiss1^ARH neurons, and elevated firing of Kiss1^ARH neurons stimulates the release of both NKB and dynorphin³². While NKB binds to TacR3 in postsynaptic Kiss1^ARH neurons to open TRPC 5 channels that robustly depolarizes these cells, contemporaneously released dynorphin activates presynaptic κ-opioid receptors to limit the release of NKB to discrete bursts of activity³². Thus, these two peptide neurotransmitters coordinate the synchronous firing of Kiss1^ARH neurons that governs the pulsatile release of GnRH into the median eminence^32,34,37. Recent computational modeling has verified that TRPC 5 and GIRK channels are critical for synchronized firing similar to what has been described for lateral septal neurons³⁸.

The amplitude of the slow EPSP and hence the synchronous firing of Kiss1-^ARH neurons is significantly greater in the ovariectomized females³², which ultimately drives the pulsatile release of LH from the pituitary gland³⁹. With rising serum levels of E2, the pulse generator activity is slowed, and in vitro we observe a reduced amplitude of the slow EPSP that recruits Kiss1^ARH neurons for synchronous firing. Indeed, E2 significantly down-regulates the expression of Trpc5 and Girk2 mRNA in Kiss1^ARH neurons, which results in the attenuated slow EPSP. However, there appears to be a physiological transitioning of Kiss1^ARH neurons from synchronous firing to burst firing for releasing glutamate that facilitates the preovulatory surge of GnRH and LH^40,41.

A12 Dopamine neurons and control of prolactin secretion

The anterior pituitary secretion of prolactin is under direct inhibitory control by hypothalamic tuberoinfundibular (A12) dopamine neurons. Dopamine is released from their axon terminals of the A12 dopamine neurons into the median eminence portal circulation and is transported to the anterior pituitary gland, where it inhibits the secretion of prolactin from pituitary lactotrophs via its Gi/o-coupled D2 receptor-mediated opening of GIRK channels⁴². Precise control of prolactin secretion by the A12 dopamine neurons is essential for successful reproduction, maternal behavior, sexual drive, metabolism, and even immune system function⁴³. During early pregnancy, a decline in portal blood dopamine results in an increase in serum prolactin levels, thereby preparing the uterine endometrium for implantation of a fertilized ovum⁴⁴. Equally important physiologically is the suckling-induced decrease in dopamine release in the postpartum female, which results in increased serum prolactin levels. There is a tight correlation between the firing activity of the A12 dopamine neurons and dopamine release into the median eminence^45,46. The A12 dopamine neurons show a rhythmic “oscillatory” firing behavior that transitions to a tonic firing mode with the excitatory input from Thyrotropin-releasing hormone (TRH) neurons⁴⁷; similar effects are seen with short-loop feedback of circulating prolactin, which is released by pituitary lactotrophs⁴⁸. A more recent paper has revealed that TRPC 5 channels generate a plateau potential with tonic firing in A12 dopamine neurons in response to prolactin⁴⁹. Indeed, conditional knockout of TRPC 5 channels in dopamine neurons abrogates the prolactin-induced plateau potential and tonic firing. Therefore, the findings by Blum and colleagues are consistent with the results in Kiss1^ARH neurons that the activation of TRPC5 channels underlies the slow EPSP (plateau potential) and sustained firing.

TRPC channel signaling in homeostatic energy circuits

POMC neurons and energy balance

POMC neurons and their orexigenic NPY/AgRP counterparts are primary CNS targets of both insulin and leptin^10,50–52. It was originally found that the anorexigenic hormone leptin depolarizes and thereby excites POMC neurons by opening non-selective cation channels⁵³; some years later it was discovered that TRPC 5 channels are in fact the non-selective cation channels underlying this response^{9,10,15,54,55}. Likewise, insulin depolarizes POMC neurons via activation of TRPC 5 channels, and both hormones hyperpolarize and thereby inhibit orexigenic NPY/AgRP neurons by opening K_ATP channels^10,56. These contrasting cellular but physiologically congruent effects of leptin and insulin dictate their anorexigenic actions. The increased POMC excitability caused by insulin translates into heightened transcriptional activity—i.e., increased c-Fos expression in the arcuate nucleus and specifically in POMC neurons after icv insulin¹⁰. Insulin delivered directly into the third ventricle consistently decreases food intake in guinea pigs¹⁰, mice^57,58 and rats⁵⁹. The insulin-induced reduction in food intake coincides with altered energy expenditure as manifested by increased O₂ consumption, CO₂ production and metabolic heat production¹⁰. Melanocortin receptor 3, 4 antagonists abrogate the catabolic effects of insulin, which indicates that the actions of insulin are mediated by POMC neurons⁵⁷. Optogenetic and chemogenetic stimulation of NPY/AgRP neurons rapidly augments food consumption^60,61, and optogenetic stimulation of POMC neurons lowers food intake albeit more slowly^60,62. The actions of leptin and insulin in POMC neurons are critical for both the short term (excitability) and long-term (transcriptional) regulation of POMC excitability as well as the control of food intake and ultimately energy homeostasis.

Insulin resistance (IR) is a central feature of the metabolic syndrome and associated with deficient insulin signaling in cells throughout the body. Neurons, like fat and muscle cells, can develop hyperinsulinemia-induced IR; resulting in severe insults to the nervous system as seen in diabetic neuropathies⁶³. Moreover, males are more susceptible to the metabolic syndrome than women in early adult life, but this sex difference lessens considerably in hypo-estrogenic states^64,65. Rodent exposure to a high fat diet over 6–10 weeks invariably triggers diet-induced obesity (DIO) and dramatic maladaptations in multiple physiological systems. The DIO model has been utilized for electrophysiological investigations of NPY/AgRP and POMC neurons^66–70. DIO Pomc^EGFP mice have been used for thorough cellular and molecular studies in order to elucidate deficits in the insulin-signaling pathway^27,71. In DIO males there is a significant diminution in the insulin-induced activation of TRPC5 channels in POMC neurons that can be attributed to an upregulation of T-cell protein tyrosine phosphatase (TCPTP), which decouples InsRs from TRPC 5 channel opening⁷¹. Supporting this idea is the fact that the thiazolidinedione rosiglitazone⁷² fully activates these channels in POMC neurons from DIO males²⁷. Therefore, TRPC 5 channels are clearly not downregulated in POMC neurons from DIO males, but instead there is an uncoupling of insulin receptors from their effector TRPC 5 channels.

There is an appreciable sex disparity in the response to insulin with DIO. Similar to the males, no differences in the resting membrane potential or input resistance of POMC neurons are observed between control and DIO female mice; but in contrast to males, the steady-state response (inward current) to insulin is not decremented with DIO²⁷. The reversal potential for the inward current caused by insulin is similar both for control-fed and DIO females and as expected the insulin response is negated by the TRPC channel blocker 2-APB⁴. Moreover, insulin robustly depolarizes and increases the firing rate of POMC neurons from DIO females similar to that seen in chow-fed controls. Hence, there is no attenuation of the insulin-induced activation of TRPC 5 channels in POMC neurons from DIO, proestrous females, indicating that there is a clear sex difference in the pathogenesis of insulin resistance in POMC neurons.

In contrast to gonadally intact, proestrous females, POMC neurons from ovariectomized, DIO females are completely refractory to insulin’s effects²⁷. However, the TRPC 5 channel opener rosiglitazone still elicits a robust inward current, which demonstrates that TRPC 5 channels are expressed and functional in POMC neurons from DIO, ovariectomized females. Conversely, E2 administered to ovariectomized, DIO females restores the sensitivity of POMC neurons to the insulin response—i.e., insulin induces a robust inward current and depolarizes POMC neurons. Therefore, in the absence of E₂ there is an uncoupling of InsRs from their effector TRPC 5 channels in obese females, but the insulin response is rescued with E₂ replacement.

Importantly, deletion of both insulin and leptin receptors in POMC neurons causes overt systemic insulin resistance in both male and female mice⁷³. If left unchecked, insulin-deficiency leads to hyperglycemia, polyuria, ketoacidosis and death as seen in type 1 diabetes. However, insulin-deficient rodents are sustained with leptin monotherapy, and this life-saving therapy is efficacious in mice expressing leptin receptors only in hypothalamic POMC and GABAergic neurons⁷⁴. Therefore, Coppari and colleagues⁷⁴ stipulated that leptin and insulin must engage the same hypothalamic circuitry to maintain glucose homeostasis and hepatic function, which at the cellular level means that insulin and leptin converge upon a common effector like TRPC 5 channels to excite POMC neurons to maintain homeostatic function¹⁰. Additionally, TRPC 3 channels may contribute to the glucose-mediated excitation of neurons in the hypothalamic ventromedial nucleus (VMH; Table 1)⁷⁵, and therefore provide further excitatory drive to POMC neurons^76,77.

Table 1.

Summary of TRPC channel subtype expression and their cell-type dependent effects

CELL TYPE	EXPRESSION LEVEL OF TRPC CHANNEL	EFFECT ON CELL	STIMULATOR	PHYSIOLOGICAL FUNCTION	REFERENCE
GnRH neurons (m)	4>1>5	Increases excitability	Kisspeptin	Reproduction	101,102,103
POMC neurons (m, g)	5>1>4>7>6 (m)	Increases excitability	Leptin, insulin, PACAP, GLP1	Energy homeostasis; anorexigenic	9(m),10(g),55(m),99(m) 79(m,g)
ARH Kiss1 neurons (m,g)	5 (m)	Increases excitability	Senktide, insulin	Reproduction	16(m),10(g) 104(m)
Hypocretin/Orexin neurons (r)	4,5>7>1>3	Increases excitability	Unknown, but controls excitability	Arousal	105
Vasopressin neurons (r)	4	Increases excitability	Water deprivation	Body fluid homeostasis	106
Glutamatergic MnPO neurons (m)	1,5,7; PCR only	Increase excitability	Histamine	Thermoregulation	107
Glucose-excited neurons in MBH (m)	3	Increases excitability	Glucose	Energy homeostasis	75
PMV neurons (m)	Putative, pharmacology only	Increases excitability	Leptin	Reproduction	108
GABAergic neurons in LH (m)	Putative, pharmacology only	Increases excitability results in MCH neuron inhibition	TRH	Anorexic and arousal actions	109

Abbreviations: ARH, arcuate nucleus; GnRH, gonadotropin- releasing hormone; LH, lateral hypothalamus; MBH, mediobasal hypothalamus; MCH, melanin-concentrating hormone; MnPO, median preoptic nucleus; PCR, polymerase chain reaction; PMV, ventral premammillary nucleus; POMC, pro-opiomelanocortin; TRH, thyrotropin-releasing hormone. (m), mouse; (r), rat; (g), guinea pig.

Similar to Kiss1^ARH neurons, TRPC 5 channels are also an effector target for multiple peptide receptors in POMC neurons. One prime example is pituitary adenylyl cyclase-activating polypeptide (PACAP). PACAP can exert both orexigenic and anorexigenic actions, depending on energy status and the population of neurons synthesizing and secreting the peptide^78,79,80,81. In the VMH, PACAP is co-expressed in the vast majority of neurons containing steroidogenic factor (SF)1, a transcription factor that upregulates PACAP expression in these cells^82,83,79. Global SF1 knockout mice develop obesity as early as eight weeks of age⁸⁴. Additionally, Cre-dependent apoptotic lesioning of VMH PACAP neurons accelerates weight gain, increases energy intake and meal frequency and reduces energy expenditure in chow-fed PACAP^Cre mice. These effects are wholly accentuated in HFD-fed males but either completely blunted (in the case of energy intake and meal frequency) or altogether reversed (in the case of weight gain and energy expenditure) in HFD-fed females⁸¹. VMH SF1/PACAP neurons are glutamatergic, and project to the ARH and synapse on POMC neurons^85,86. Transient optogenetic stimulation of VMN SF1/PACAP neurons elicits a fast excitatory postsynaptic potential (EPSP) in POMC neurons^77,87, whereas PACAP, which is released with more sustained photostimulation (20 Hz), generates a slow EPSP that depolarizes and increases the firing rate of POMC neurons^79,80,81. These effects are abrogated by either pharmacological blockade or genetic ablation of its cognate PAC1 receptor, as well as by blockade of TRPC 5 channels^79,80,81. The PAC1 receptor works through both G_q- and PI3K –mediated signaling to elicit this excitation of POMC neurons⁷⁹. These cellular actions account, at least in part, for the PACAP-induced suppression of energy intake an increase in energy expenditure^79,80,81. The PAC1 receptor/TRPC 5 channel coupling in POMC neurons is potentiated by E2 in females and attenuated by DIO in males^79,80,81. Interestingly, the polarity of the PACAP response in these cells flips from excitatory to inhibitory under fasting conditions as the PAC1 receptor/effector coupling switches from TRPC 5 to ATP-gated K⁺ channels. This is evidenced by the fact that the PACAP-induced inward current, depolarization and increase in firing seen in POMC neurons under ad libitum-fed conditions switches to an outward current, hyperpolarization and suppression of firing under conditions of negative energy balance brought on by five consecutive days of an 18-hour fast/six-hour refeed paradigm⁸⁰. These fasting-induced alterations are attributed to increases in the expression and activity of AMP-dependent protein kinase (AMPK) and TCPTP - energy sensing signaling molecules that also regulate the coupling of leptin, insulin and ghrelin receptors with their effector systems^88,89,90,80. Indeed, the PACAP-induced inhibition of POMC neurons under fasting conditions rapidly reverts back to an excitatory response in the presence of inhibitors of AMPK and TCPTP and is mimicked under ad libitum conditions upon co-administration of AMPK activators⁸⁰. Therefore, the coupling of peptide and metabolic hormone receptors to TRPC 5 channels is exquisitely regulated in different physiological states.

Emerging evidence also indicates that glucagon-like peptide (GLP) 1 activates TRPC 5 channels. This incretin is secreted postprandially from intestinal L cells in response to neural stimulation from the vagus and nutrient signals in the lumen, which results in enhanced glucose-dependent insulin secretion⁹¹ thereby ameliorating the clinical impact of obesity and associated comorbidities⁹². GLP1 and its analogues decrease both homeostatic and hedonic feeding^93,94,95. GLP1 receptors are found in hypothalamic areas like the ARH, paraventricular nucleus of the hypothalamus (PVH) and VMH⁹⁶; and GLP1 neurons in the nucleus tractus solitarius project to areas sub-serving both homeostatic and hedonic energy balance circuits^97,95. The expression of GLP1 receptors in POMC neurons exhibits a distinct yet overlapping distribution compared to that of leptin receptors, although this difference in receptor expression is not apparent in GABAergic POMC neurons⁹⁸. These GLP1 receptor-expressing POMC neurons have a differing array of intrinsic conductances and are more inherently excitable⁹⁸. In addition, a greater percentage of them respond to GLP1 with depolarization and increased firing than leptin receptor-expressing POMC neurons do in response to leptin⁹⁸. Moreover, the GLP1-induced increase in excitability is replicated by its liraglutide analogue⁹⁹. Furthermore, selective knockdown of TRPC 5 channels in POMC neurons nullifies the depolarizing effect of liraglutide as well as the anorexigenic effect caused by systemic injection⁹⁹.

Finally, serotoninergic projections from the brainstem dorsal raphe nucleus inhibit hunger-driven feeding in mice¹⁰⁰, and serotonin binds to its 5HT_2C receptor (Gq-coupled) to activate TRPC 5 channels in POMC neurons¹⁵. Deletion of TRPC 5 channels specifically in POMC neurons results in a decrease in energy expenditure and increase in food intake and weight gain in male mice⁵⁵. Therefore, TRPC channels in POMC neurons are a conduit for mediating the effects of monoamine, peptide neurotransmitter and metabolic hormones on the excitability and subsequently the hypothalamic control of energy homeostasis.

Conclusions

TRPC channels were identified in the early part of the century as a major target for group I metabotropic glutamate receptor (mGluR1) signaling in CNS neurons^8,12–14. Subsequently, TPRC channels were identified as key players involved in hypothalamic control of multiple homeostatic functions (Table 1). As such they are activated by peptide G protein-coupled receptors similar to other CNS structures (see^1,11 for review) and in addition to metabolic hormone receptors (InsR and LRb), which can occur in the same neuron. In Kiss1^ARH neurons, for example, leptin and insulin through their cognate receptors signal via PI3K to activate TRPC 5 channels in different metabolic states^10,54, whereas the Gαq-coupled TacR3 activates TRPC 5 channels in reproductive states³². Likewise, in POMC neurons the PAC1 receptor is Gαq-coupled to activate TRPC 5 channels^79,80,81. In DIO males (and fasted subjects as well) the receptors are uncoupled from TRPC 5 channel opening through the activity of AMPK and TCPTP. Furthermore, the importance of preserving this critical metabolic signaling pathway is highlighted by the fact that E₂ is able to protect females from the development of insulin resistance through preserving TRPC 5 channel coupling (and Trpc5 mRNA expression). Therefore, TRPC 5 channel activity is highly regulated in both physiological and pathological states. A future challenge will be to use specific genetic tools (e.g., sgRNA’s) for probing TRPC channel function in different physiologic and pathophysiologic states.