Kevin P. M. Currie

Regulation of CaV2 calcium channels by G protein coupled receptors

Voltage gated calcium channels (Ca²⁺ channels) are key mediators of depolarization induced calcium influx into excitable cells, and thereby play pivotal roles in a wide array of physiological responses. This review focuses on the inhibition of Ca(V)2 (N- and P/Q-type) Ca²⁺-channels by G protein coupled receptors (GPCRs), which exerts important autocrine/paracrine control over synaptic transmission and neuroendocrine secretion. Voltage-dependent inhibition is the most widespread mechanism, and involves direct binding of the G protein βγ dimer (Gβγ) to the α1 subunit of Ca(V)2 channels. GPCRs can also recruit several other distinct mechanisms including phosphorylation, lipid signaling pathways, and channel trafficking that result in voltage-independent inhibition. Current knowledge of Gβγ-mediated inhibition is reviewed, including the molecular interactions involved, determinants of voltage-dependence, and crosstalk with other cell signaling pathways. A summary of recent developments in understanding the voltage-independent mechanisms prominent in sympathetic and sensory neurons is also included. This article is part of a Special Issue entitled: Calcium channels.

Calcium‐activated currents in cultured neurones from rat dorsal root ganglia

1 Voltage‐activated Ca2+ currents and caffeine (1 to 10 mm) were used to increase intracellular Ca2+ in rat cultured dorsal root ganglia (DRG) neurones. Elevation of intracellular Ca2+ resulted in activation of inward currents which were attenuated by increasing the Ca2+ buffering capacity of cells by raising the concentration of EGTA in the patch solution to 10 mm. Low and high voltage‐activated Ca2+ currents gave rise to Cl− tail currents in cells loaded with CsCl patch solution. Outward Ca2+ channel currents activated at very depolarized potentials (Vc + 60 mV to + 100 mV) also activated Cl− tail currents, which were enhanced when extracellular Ca2+ was elevated from 2 mm to 4 mm. 2 The Ca2+‐activated Cl− tail currents were identified by estimation of tail current reversal potential by use of a double pulse protocol and by sensitivity to the Cl− channel blocker 5‐nitro 2‐(3‐phenylpropylamino) benzoic acid (NPPB) applied at a concentration of 10 μm. 3 Cells loaded with Cs acetate patch solution and bathed in medium containing 4 mm Ca2+ also had prolonged Ca2+‐dependent tail currents, however these smaller tail currents were insensitive to NPPB. Release of Ca2+ from intracellular stores by caffeine gave rise to sustained inward currents in cells loaded with Cs acetate. Both Ca2+‐activated tail currents and caffeine‐induced inward currents recorded from cells loaded with Cs acetate were attenuated by Tris based recording media, and had reversal potentials positive to 0 mV suggesting activity of Ca2+‐activated cation channels. 4 Our data may reflect (a) different degrees of association between Ca2+‐activated channels with voltage‐gated Ca2+ channels, (b) distinct relationships between channels and intracellular Ca2+ stores and Ca2+ homeostatic mechanisms, (c) regulation of Ca2+‐activated channels by second messengers, and (d) varying channel sensitivity to Ca2+, in the cell body of DRG neurones.

Activation of Ca(2+)-dependent Cl- currents in cultured rat sensory neurones by flash photolysis of DM-nitrophen.

1. Voltage‐gated Ca2+ currents (ICa) and Ca(2+)‐activated Cl‐ currents (ICl(Ca)) were recorded from cultured rat dorsal root ganglion (DRG) neurones using the whole‐cell configuration of the patch clamp technique. Intracellular photorelease of Ca2+ by flash photolysis of DM‐nitrophen elicited transient inward currents only in those cells which possessed Ca(2+)‐activated Cl‐ tail currents following ICa. The reversal potential of the flash responses was hyperpolarized when extracellular Cl‐ was replaced by SCN‐. The flash responses and the Ca(2+)‐activated Cl‐ tail currents were inhibited by the Cl‐ channel blockers niflumic acid (10‐100 microM) and 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) (10 microM). 2. After activation by ICa, the Ca(2+)‐activated Cl‐ current could be reactivated during its decay by photorelease of caged Ca2+. Experiments carried out on neurones held at 0 mV demonstrated that ICl(Ca) could be chronically activated due to residual Ca2+ influx. These data directly demonstrated that the decay of ICl(Ca) is not due to inactivation but rather to deactivation as a result of removal of the Ca2+ load from the cell cytoplasm. 3. Photorelease of caged inositol 1,4,5‐trisphosphate (IP3) failed to activate any Ca(2+)‐dependent current responses in cultured DRG neurones, although application of caffeine elicited transient inward currents, and responses to photoreleased IP3 could be obtained from freshly dissociated smooth muscle cells. 4. Photorelease of Ca2+ provides a useful method for investigating the properties of ICl(Ca) independently from other physiological parameters. In addition, we have directly demonstrated that ICl(Ca) in DRG neurones does not inactivate, and so may continue to modulate membrane excitability as long as the intracellular Ca2+ concentration ([Ca2+]i) close to the cell membrane is elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential facilitation of N- and P/Q-type calcium channels during trains of action potential-like waveforms

Inhibition of presynaptic voltage‐gated calcium channels by direct G‐protein βγ subunit binding is a widespread mechanism that regulates neurotransmitter release. Voltage‐dependent relief of this inhibition (facilitation), most likely to be due to dissociation of the G‐protein from the channel, may occur during bursts of action potentials. In this paper we compare the facilitation of N‐ and P/Q‐type Ca2+ channels during short trains of action potential‐like waveforms (APWs) using both native channels in adrenal chromaffin cells and heterologously expressed channels in tsA201 cells. While both N‐ and P/Q‐type Ca2+ channels exhibit facilitation that is dependent on the frequency of the APW train, there are important quantitative differences. Approximately 20 % of the voltage‐dependent inhibition of N‐type ICa was reversed during a train while greater than 40 % of the inhibition of P/Q‐type ICa was relieved. Changing the duration or amplitude of the APW dramatically affected the facilitation of N‐type channels but had little effect on the facilitation of P/Q‐type channels. Since the ratio of N‐type to P/Q‐type Ca2+ channels varies widely between synapses, differential facilitation may contribute to the fine tuning of synaptic transmission, thereby increasing the computational repertoire of neurons.

G protein inhibition of CaV2 calcium channels

Voltage-gated Ca2+ channels translate the electrical inputs of excitable cells into biochemical outputs by controlling influx of the ubiquitous second messenger Ca2+. As such the channels play pivotal roles in many cellular functions including the triggering of neurotransmitter and hormone release by CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels. It is well established that G protein coupled receptors (GPCRs) orchestrate precise regulation neurotransmitter and hormone release through inhibition of CaV2 channels. Although the GPCRs recruit a number of different pathways, perhaps the most prominent, and certainly most studied among these is the so-called voltage-dependent inhibition mediated by direct binding of Gβγ to the α1 subunit of CaV2 channels. This article will review the basics of Ca2+-channels and G protein signaling, and the functional impact of this now classical inhibitory mechanism on channel function. It will also provide an update on more recent developments in the field, both related to functional effects and crosstalk with other signaling pathways, and advances made toward understanding the molecular interactions that underlie binding of Gβγ to the channel and the voltage-dependence that is a signature characteristic of this mechanism.

Inhibition of Ca2+ Channels and Adrenal Catecholamine Release by G Protein Coupled Receptors

Catecholamines and other transmitters released from adrenal chromaffin cells play central roles in the “fight-or-flight” response and exert profound effects on cardiovascular, endocrine, immune, and nervous system function. As such, precise regulation of chromaffin cell exocytosis is key to maintaining normal physiological function and appropriate responsiveness to acute stress. Chromaffin cells express a number of different G protein coupled receptors (GPCRs) that sense the local environment and orchestrate this precise control of transmitter release. The primary trigger for catecholamine release is Ca2+ entry through voltage-gated Ca2+ channels, so it makes sense that these channels are subject to complex regulation by GPCRs. In particular G protein βγ heterodimers (Gβγ) bind to and inhibit Ca2+ channels. Here I review the mechanisms by which GPCRs inhibit Ca2+ channels in chromaffin cells and how this might be altered by cellular context. This is related to the potent autocrine inhibition of Ca2+ entry and transmitter release seen in chromaffin cells. Recent data that implicate an additional inhibitory target of Gβγ on the exocytotic machinery and how this might fine tune neuroendocrine secretion are also discussed.

Molecular Characterization of the Gerbil C5a Receptor and Identification of a Transmembrane Domain V Amino Acid That Is Crucial for Small Molecule Antagonist Interaction

Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.

G-Proteins Modulate Cumulative Inactivation of N-Type (CaV2.2) Calcium Channels

Precise regulation of N-type (CaV2.2) voltage-gated calcium channels (Ca-channels) controls many cellular functions including neurotransmitter and hormone release. One important mechanism that inhibits Ca2+ entry involves binding of G-protein βγ subunits (Gβγ) to the Ca-channels. This shifts the Ca-channels from “willing” to “reluctant” gating states and slows activation. Voltage-dependent reversal of the inhibition (facilitation) is thought to reflect transient dissociation of Gβγ from the Ca-channels and can occur during high-frequency bursts of action potential-like waveforms (APW). Inactivation of Ca-channels will also limit Ca2+ entry, but it remains unclear whether G-proteins can modulate inactivation. In part this is because of the complex nature of inactivation, and because facilitation of Ca-channel currents (ICa) masks the extent and kinetics of inactivation during typical stimulation protocols. We used low-frequency trains of APW to activate ICa. This more closely mimics physiological stimuli and circumvents the problem of facilitation which does not occur at ≤5 Hz. Activation of endogenous G-proteins reduced both Ca2+-dependent, and voltage-dependent inactivation of recombinant ICa in human embryonic kidney 293 cells. This was mimicked by expression of wild-type Gβγ, but not by a point mutant of Gβγ with reduced affinity for Ca-channels. A similar decrease in the inactivation of ICa was produced by P2Y receptors in adrenal chromaffin cells. Overall, our data identify and characterize a novel effect of G-proteins on ICa, and could have important implications for understanding how G-protein-coupled receptors control Ca2+ entry and Ca2+-dependent events such as neurotransmitter and hormone release.

Electrochemical detection of catecholamine release using planar iridium oxide electrodes in nanoliter microfluidic cell culture volumes

Release of neurotransmitters and hormones by calcium regulated exocytosis is a fundamental cellular/molecular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. Therefore, this area represents a relevant target for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistically rich data with increased throughput. Toward this goal, we have electrochemically deposited iridium oxide (IrOx) films onto planar thin film platinum electrodes (20 μm×300 μm) and utilized these for quantitative detection of catecholamine release from adrenal chromaffin cells trapped in a microfluidic network. The IrOx electrodes show a linear response to norepinephrine in the range of 0-400 μM, with a sensitivity of 23.1±0.5 mA/M mm(2). The sensitivity of the IrOx electrodes does not change in the presence of ascorbic acid, a substance commonly found in biological samples. A replica molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. Small populations of chromaffin cells were trapped in the microfluidic device and stimulated by rapid perfusion with high potassium (50mM) containing Tyrodes solution at a flow rate of 1 nL/s. Stimulation of the cells produced a rapid increase in current due to oxidation of the released catecholamines, with an estimated maximum concentration in the cell culture volume of ~52 μM. Thus, we demonstrate the utility of an integrated microfluidic network with IrOx electrodes for real-time quantitative detection of catecholamines released from small populations of chromaffin cells.

N- and P/Q-type Ca2+ channels in adrenal chromaffin cells.

Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage‐dependent Ca2+ channels, found in all excitable cells ( Hille 1992 ), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore‐forming subunit, called the α1 subunit, and several accessory subunits. Ten different α1 subunit genes have been identified and classified into three families, Cav1‐3 ( Dunlap et al. 1995 , Catterall 2000 ). Each α1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav2.1 and Cav2.2 channels, also known as P/Q‐ and N‐type respectively ( Nowycky et al. 1985 , Llinas et al. 1989b , Wheeler et al. 1994 ). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N‐, P/Q and to a lesser extent R‐type Ca2+ channels are known to regulate neurotransmitter release ( Hirning et al. 1988 , Horne & Kemp 1991 , Uchitel et al. 1992 , Luebke et al. 1993 , Takahashi & Momiyama 1993 , Turner et al. 1993 , Regehr & Mintz 1994 , Wheeler et al. 1994 , Wu & Saggau 1994 , Waterman 1996 , Wright & Angus 1996 , Reid et al. 1997 ). N‐ and P/Q‐type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells ( Garcia et al. 2006 ). N‐ and P/Q‐type channels are subject to many forms of regulation ( Ikeda & Dunlap 1999 ). This review pays particular attention to the regulation of N‐ and P/Q‐type channels by heterotrimeric G‐proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus‐secretion coupling in adrenal chromaffin cells.