Patch Clamp Analysis of Opioid-Induced Kir3 Currents in Mouse Peripheral Sensory Neurons Following Nerve Injury.

Patch clamp is an electrophysiological technique that allows to analyze the activity of ion channels in neurons. In this chapter, we provide a detailed description of patch clamp protocol to measure the effect of a µ-opioid receptor agonist on the activity of G protein-coupled inwardly rectifying potassium (GIRK or Kir3) channels. This is performed in peripheral sensory neurons isolated from dorsal root ganglia (DRG) of mice without or with a chronic constriction injury (CCI) of the sciatic nerve, which models neuropathic pain. We describe the induction of the CCI , isolation and culture of DRG neurons, performance of the patch clamp recordings, and identification of opioid-responding neurons.

A Regional Reduction in Ito and IKACh in the Murine Posterior Left Atrial Myocardium Is Associated with Action Potential Prolongation and Increased Ectopic Activity.

BACKGROUND: The left atrial posterior wall (LAPW) is potentially an important area for the development and maintenance of atrial fibrillation. We assessed whether there are regional electrical differences throughout the murine left atrial myocardium that could underlie regional differences in arrhythmia susceptibility. METHODS: We used high-resolution optical mapping and sharp microelectrode recordings to quantify regional differences in electrical activation and repolarisation within the intact, superfused murine left atrium and quantified regional ion channel mRNA expression by Taqman Low Density Array. We also performed selected cellular electrophysiology experiments to validate regional differences in ion channel function. RESULTS: Spontaneous ectopic activity was observed during sustained 1Hz pacing in 10/19 intact LA and this was abolished following resection of LAPW (0/19 resected LA, P<0.001). The source of the ectopic activity was the LAPW myocardium, distinct from the pulmonary vein sleeve and LAA, determined by optical mapping. Overall, LAPW action potentials (APs) were ca. 40% longer than the LAA and this region displayed more APD heterogeneity. mRNA expression of Kcna4, Kcnj3 and Kcnj5 was lower in the LAPW myocardium than in the LAA. Cardiomyocytes isolated from the LAPW had decreased Ito and a reduced IKACh current density at both positive and negative test potentials. CONCLUSIONS: The murine LAPW myocardium has a different electrical phenotype and ion channel mRNA expression profile compared with other regions of the LA, and this is associated with increased ectopic activity. If similar regional electrical differences are present in the human LA, then the LAPW may be a potential future target for treatment of atrial fibrillation.

Role for germline mutations and a rare coding single nucleotide polymorphism within the KCNJ5 potassium channel in a large cohort of sporadic cases of primary aldosteronism.

Primary aldosteronism (autonomous aldosterone production with suppressed renin) plays an important pathophysiological role in what has been previously labeled as essential hypertension. Besides the recently described germline mutations in the KCNJ5 potassium channel associated with familial primary aldosteronism, somatic mutations in the same channel have been identified within aldosterone-producing adenomas. In this study, we have resequenced the flanking and coding region of KCNJ5 in peripheral blood DNA from 251 white subjects with primary aldosteronism to look for rare variants that might be important for the pathophysiology of sporadic primary aldosteronism. We have identified 3 heterozygous missense mutations (R52H, E246K, and G247R) in the cohort and found that 12 (5% of the cohort) were carriers for the rare nonsynonymous single nucleotide polymorphism rs7102584 causing E282Q substitution of KCNJ5. By expressing the channels in Xenopus oocytes and human adrenal H295R cells, we have shown that the R52H, E246K, and E282Q substitutions are functional, but the G247R mutation is indistinguishable from wild type. Although the functional substitutions are remote from the selectivity filter, they affect the inward-rectification, the ability of the KCNJ5 channels to conduct Na(+) currents and ATII-induced aldosterone release from the H295R cell line. Together these data suggest that germline variation in the KCNJ5 gene has a role to play in the common sporadic form as well as the much rarer syndromic forms of primary aldosteronism.

Differential maturation of GIRK2-expressing neurons in the mouse cerebellum.

The cerebellar cortex is among the brain regions showing the highest expression levels of G-protein-gated inwardly-rectifying potassium (GIRK/Kir3) channels. Despite their critical contribution in modulating neuronal excitability during development and adult, the spatiotemporal expression of specific GIRK subunits in identified cerebellar neuron populations is unresolved. To characterize this onset of expression, we examined the GIRK2 protein expression in mouse cerebellum by western blot, light microscopy immunohistochemistry and immunofluorescence during perinatal development. Using western blots, GIRK2 expression was low at birth but reach its maximum at P5 before decreasing gradually to adult levels. Immunohistochemical localization indicated that GIRK2 is expressed in granule cells from early stages of development. At the embryonic stage, immunofluorescence techniques for the transcription factor Pax6 allowed to demonstrate that GIRK2 is expressed in granule cell precursors. This GIRK2 expression in granule cells continued throughout postnatal development and adulthood. In addition, the expression of Pax2-GFP allowed selective visualization of Golgi cells during pre- and postnatal development. We could not detect co-expression of Pax2-GFP and GIRK2 during prenatal and early postnatal development, but only at post-migratory stages of Golgi cells, once they are morphologically differentiated and located at the granule cell layer. In the adult cerebellum, we performed a detailed characterization on the expression of GIRK2 in different subpopulations of Golgi cells, using metabotropic glutamate receptor 2 (mGlu(2)) and neurogranin as markers, in GlyT2-GFP and GAD67-GFP mice, and showed that GIRK2 is present in at least four morphological and neurochemical non-overlapping populations of Golgi cells. Altogether, these findings shed new light on the developmental regulation of GIRK channels in the cerebellum, and the main expression in granule cells during perinatal development support the idea that GIRK2 may provide a significant route for modulating different aspects of cerebellar development.

A fluorescent screening assay for identifying modulators of GIRK channels.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue(1,2). GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate. GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders(3). However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations(3). Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry(4) or radiotracer analysis(5), the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

Silencing GIRK4 expression in human atrial myocytes by adenovirus-delivered small hairpin RNA.

GIRK4 has been shown to be a subunit of I(KACh), and the use of GIRK4 in human atrial myocytes to treat arrhythmia remains an important research pursuit. Adenovirus-delivered small hairpin RNA (shRNA) has been used to mediate gene knockdown in mouse cardiocytes, yet there is no information on the successful application of this technique in human cardiocytes. In the current study, we used a siRNA validation system to select the most efficient sequence for silencing GIRK4. To this end, adenovirus-delivered shRNA, which expresses this sequence, was used to silence GIRK4 expression in human atrial myocytes. Finally, the feasibility, challenges, and results of silencing GIRK4 expression were evaluated by RT-PCR, western blotting, and the voltage-clamp technique. The levels of mRNA and protein were depressed significantly in cells infected by adenovirus-delivered shRNA against GIRK4, approximately 86.3% and 51.1% lower than those cells infected by adenovirus-delivered nonsense shRNA, respectively. At the same time, I(KACh) densities were decreased 53% by adenovirus-delivered shRNA against GIRK4. In summary, adenovirus-delivered shRNA against GIRK4 mediated efficient GIRK4 knockdown in human atrial myocytes and decreased I(KACh) densities. As such, these data indicated that adenovirus-delivered shRNA against GIRK4 is a potential tool for treating arrhythmia.

Changes in I K, ACh single-channel activity with atrial tachycardia remodelling in canine atrial cardiomyocytes.

AIMS: Although atrial tachycardia (AT) remodelling promotes agonist-independent, constitutively active, acetylcholine-regulated K+-current (I K,ACh) that increases susceptibility to atrial fibrillation (AF), the underlying changes in I K,Ach channel function are unknown. This study aimed to establish how AT remodelling affects I K,ACh single-channel function. METHODS AND RESULTS: I K,ACh single-channel activity was studied via cell-attached patch-clamp in isolated left atrial cardiomyocytes of control and AT (7 days, 400 min(-1)) dogs. Atrial tachycardia prolonged the mean duration of induced AF from 44 +/- 22 to 413 +/- 167 s, and reduced atrial effective refractory period at a 360 ms cycle length from 126 +/- 3 to 74 +/- 5 ms (n = 9/group, P < 0.001). In the absence of cholinergic stimulation, single-channel openings with typical I K,ACh conductance and rectification properties were sparse under control conditions. Atrial tachycardia induced prominent agonist-independent I K,ACh activity because of increased opening frequency (fo) and open probability (Po: approximately seven- and 10-fold, respectively, vs. control), but did not alter open time-constant, single-channel conductance, and membrane density. With maximum I K,ACh activation (10 micromol/L carbachol), channel Po was enhanced much more in control cells ( approximately 42-fold) than in AT-remodelled myocytes (approximately five-fold). The selective Kir3 current blocker tertiapin-Q (100 nmol/L) reduced fo and Po at -100 mV by 48 and 51%, respectively (P < 0.05 for each), without altering other channel properties, confirming the identity of I K,ACh. Atrial tachycardia had no significant effect on mRNA or protein expression of either of the subunits (Kir3.1, Kir3.4) underlying I K,ACh. CONCLUSION: Atrial tachycardia increases agonist-independent constitutive I K,ACh single-channel activity by enhancing spontaneous channel opening, providing a molecular basis for AT effects on macroscopic I K,ACh observed in previous studies, as well as associated refractoriness abbreviation and tertiapin-suppressible AF promotion. These results suggest an important role for constitutive I K,Ach channel opening in AT remodelling and support its interest as a potential target for AF therapy.

Compartment-dependent colocalization of Kir3.2-containing K+ channels and GABAB receptors in hippocampal pyramidal cells.

G-protein-coupled inwardly rectifying K+ channels (Kir3 channels) coupled to metabotropic GABAB receptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABAB receptors on hippocampal pyramidal cells, we used a high-resolution immunocytochemical approach. Immunoreactivity for the Kir3.2 subunit was most abundant postsynaptically and localized to the extrasynaptic plasma membrane of dendritic shafts and spines of principal cells. Quantitative analysis of immunogold particles for Kir3.2 revealed an enrichment of the protein around putative glutamatergic synapses on dendritic spines, similar to that of GABA(B1). Consistent with this observation, a high degree of coclustering of Kir3.2 and GABA(B1) was revealed around excitatory synapses by the highly sensitive SDS-digested freeze-fracture replica immunolabeling. In contrast, in dendritic shafts receptors and channels were found to be mainly segregated. These results suggest that Kir3.2-containing K+ channels on dendritic spines preferentially mediate the effect of GABA, whereas channels on dendritic shafts are likely to be activated by other neurotransmitters as well. Thus, Kir3 channels, localized to different subcellular compartments of hippocampal principal cells, appear to be differentially involved in synaptic integration in pyramidal cell dendrites.