Channel-deficient Mice Research Articles

Myocardial KATP channels are critical to Ca2+ homeostasis in the metabolically stressed heart in vivo

myocardial atp-sensitive potassium (KATP) channels appear to play central roles in the adaptation of the heart to both physiological and pathological metabolic stress ([1][1], [3][2], [5][3], [7][4], [8][5], [14][6], [16][7], [21][8]). In the heart, KATP channels are thought to act as metabolic

Modified sympathetic regulation in N-type calcium channel null-mouse

To elucidate the physiological importance of neuronal (N)-type calcium channels in sympathetic controls, we analyzed N-type channel-deficient (NKO) mice. Immunoprecipitation analysis revealed increased interaction between β3 (a major accessory subunit of N-type channels) and R-type channel-forming CaV2.3 in NKO mice. R–R intervals in NKO ECG recordings were elongated and fluctuating, suggesting disturbed sympathetic tonus. N-type channel inhibitors elongated the R–R interval in control mice, whereas R-type channel blocking with SNX-482 significantly affected NKO but not control mice, indicating a compensatory role for R-type channels. Echocardiography and Langendorff heart analysis confirmed a major role for R-type channels in NKO mice. Combined, our biochemical and physiological analyses strongly suggest that the remaining sympathetic tonus in NKO mice is dependent on R-type calcium channels.

Effects of glucocorticoid receptor antagonists on allodynia and hyperalgesia in mouse model of neuropathic pain

Injury to the spinal nerves of mice induces mechanical allodynia and thermal hyperalgesia. In the injured spinal cord, the expression of glucocorticoid receptor mRNA was increased, whereas it was decreased in N-type Ca 2+-channel-deficient mice, in which neuropathic pain is eliminated. Intrathecal and intraperitoneal injection of the glucocorticoid receptor antagonist RU486 produced antinociceptive effects, whereas intracerebroventricular injection was without effect. The more selective antagonist dexamethasone 21-mesylate suppressed both mechanical allodynia and thermal hyperalgesia. These results suggest that spinal glucocorticoid receptors play an important role in neuropathic pain, and that controlling the activity of glucocorticoid receptors may be of great importance in the treatment of neuropathic pain.

Functional Roles of Ca v 1.3(α 1D ) Calcium Channels in Atria

Background— Previous data suggest that L-type Ca 2+ channels containing the Ca v 1.3(α 1D ) subunit are expressed mainly in neurons and neuroendocrine cells, whereas those containing the Ca v 1.2(α 1C ) subunit are found in the brain, vascular smooth muscle, and cardiac tissue. However, our previous report as well as others have shown that Ca v 1.3 Ca 2+ channel–deficient mice ( Ca v 1.3 −/− ) demonstrate sinus bradycardia with a prolonged PR interval. In the present study, we extended our study to examine the role of the Ca v 1.3(α 1D ) Ca 2+ channel in the atria of Ca v 1.3 −/− mice. Methods and Results— We obtained new evidence to demonstrate that there is significant expression of Ca v 1.3 Ca 2+ channels predominantly in the atria compared with ventricular tissues. Whole-cell L-type Ca 2+ currents ( I Ca,L ) recorded from single, isolated atrial myocytes from Ca v 1.3 −/− mice showed a significant depolarizing shift in voltage-dependent activation. In contrast, there were no significant differences in the I Ca,L recorded from ventricular myocytes from wild-type and null mutant mice. We previously documented the hyperpolarizing shift in the voltage-dependent activation of Ca v 1.3 compared with Ca v 1.2 Ca 2+ channel subunits in a heterologous expression system. The lack of Ca v 1.3 Ca 2+ channels in null mutant mice would result in a depolarizing shift in the voltage-dependent activation of I Ca,L in atrial myocytes. In addition, the Ca v 1.3 -null mutant mice showed evidence of atrial arrhythmias, with inducible atrial flutter and fibrillation. We further confirmed the isoform-specific differential expression of Ca v 1.3 versus Ca v 1.2 by in situ hybridization and immunofluorescence confocal microscopy. Conclusions— Using gene-targeted deletion of the Ca v 1.3 Ca 2+ channel, we established the differential distribution of Ca v 1.3 Ca 2+ channels in atrial myocytes compared with ventricles. Our data represent the first report demonstrating important functional roles for Ca v 1.3 Ca 2+ channel in atrial tissues.

Treadmill running causes significant fiber damage in skeletal muscle of KATP channel-deficient mice

Although it has been suggested that the ATP-sensitive K(+) (K(ATP)) channel protects muscle against function impairment, most studies have so far given little evidence for significant perturbation in the integrity and function of skeletal muscle fibers from inactive mice that lack K(ATP) channel activity in their cell membrane. The objective was, therefore, to test the hypothesis that K(ATP) channel-deficient skeletal muscle fibers become damaged when mice are subjected to stress. Wild-type and K(ATP) channel-deficient mice (Kir6.2(-/-) mice) were subjected to 4-5 wk of treadmill running at either 20 m/min with 0 degrees inclination or at 24 m/min with 20 degrees uphill inclination. Muscles of all wild-type mice and of nonexercised Kir6.2(-/-) mice had very few fibers with internal nuclei. After 4-5 wk of treadmill running, there was little evidence for connective tissues and mononucleated cells in Kir6.2(-/-) hindlimb muscles, whereas the number of fibers with internal nuclei, which appear when damaged fibers are regenerated by satellite cells, was significantly higher in Kir6.2(-/-) than wild-type mice. Between 5% and 25% of the total number of fibers in Kir6.2(-/-) extensor digitum longus, plantaris, and tibialis muscles had internal nuclei, and most of such fibers were type IIB fibers. Contrary to hindlimb muscles, diaphragms of Kir6.2(-/-) mice that had run at 24 m/min had few fibers with internal nuclei, but mild to severe fiber damage was observed. In conclusion, the study provides for the first time evidence 1) that the K(ATP) channels of skeletal muscle are essential to prevent fiber damage, and thus muscle dysfunction; and 2) that the extent of fiber damage is greater and the capacity of fiber regeneration is less in Kir6.2(-/-) diaphragm muscles compared with hindlimb muscles.

Gastric inhibitory polypeptide is the major insulinotropic factor in K(ATP) null mice

ATP-sensitive K(+) (K(ATP)) channels in pancreatic beta-cells are crucial in the regulation of glucose-induced insulin secretion. Recently, K(ATP) channel-deficient mice were generated by genetic disruption of Kir6.2, the pore-forming component of K(ATP) channels, but the mice still showed a significant insulin response after oral glucose loading in vivo. Gastric inhibitory polypeptide (GIP) is a physiological incretin that stimulates insulin release upon ingestion of nutrients. To determine if GIP is the insulinotropic factor in insulin secretion in K(ATP) channel-deficient mice, we generated double-knockout Kir6.2 and GIP receptor null mice and compared them with Kir6.2 knockout mice. Double-knockout mice were generated by intercrossing Kir6.2-knockout mice with GIP receptor-knockout mice. An oral glucose tolerance test, insulin tolerance test and batch incubation study of pancreatic islets were performed on double-knockout mice and Kir6.2-knockout mice. Fasting glucose and insulin levels were similar in both groups. After oral glucose loading, blood glucose levels of double-knockout mice became elevated compared with Kir6.2-knockout mice, especially at 15 min (345+/-10 mg/dl vs 294+/-20 mg/dl, P<0.05) and 30 min (453+/-20 mg/dl vs 381+/-26 mg/dl, P<0.05). The insulin response was almost completely lost in double-knockout mice, although insulin secretion from isolated islets was stimulated by another incretin, glucagon-like peptide-1 in the double-knockout mice. Double-knockout mice and Kir6.2-knockout mice were similarly insulin sensitive as assessed by the insulin tolerance test. GIP is the major insulinotropic factor in the secretion of insulin in response to glucose load in K(ATP) channel-deficient mice.

Impaired thalamic T-type calcium channel function: a possible mechanism for visceral hyperalgesia and functional abdominal pain

Impaired thalamic T-type calcium channel function: a possible mechanism for visceral hyperalgesia and functional abdominal pain

Null mutation of alpha1D Ca2+ channel gene results in deafness but no vestibular defect in mice.

Multiple Ca2+ channels confer diverse functions to hair cells of the auditory and vestibular organs in the mammalian inner ear. We used gene-targeting technology to generate alpha1D Ca2+ channel-deficient mice to determine the physiological role of these Ca2+ channels in hearing and balance. Analyses of auditory-evoked brainstem recordings confirmed that alpha1D-/- mice were deaf and revealed that heterozygous (alpha1D+/-) mice have increased hearing thresholds. However, hearing deficits in alpha1D+/- mice were manifested mainly by the increase in threshold of low-frequency sounds. In contrast to impaired hearing, alpha1D-/- mice have balance performances equivalent to their wild-type littermates. Light and electron microscope analyses of the inner ear revealed outer hair cell loss at the apical cochlea, but no apparent abnormality at the basal cochlea and the vestibule. We determined the mechanisms underlying the auditory function defects and the normal vestibular functions by examining the Ba2+ currents in cochlear inner and outer hair cells versus utricular hair cells in alpha1D+/- mice. Whereas the whole-cell Ba2+ currents in inner hair cells consist mainly of the nimodipine-sensitive current (approximately 85%), the utricular hair cells express only approximately 50% of this channel subtype. Thus, differential expression of alpha1D channels in the cochlear and utricular hair cells confers the phenotype of the alpha1D null mutant mice. Because vestibular and cochlear hair cells share common features and null deletion of several genes have yielded both deafness and imbalance in mice, alpha1D null mutant mice may serve as a model to disentangle vestibular from auditory-specific functions.

Augmentation of tone, attenuation of NO/cGMP-mediated dilation and hypertension in small resistance vessel-sized arteries of BK channel deficient mice

Augmentation of tone, attenuation of NO/cGMP-mediated dilation and hypertension in small resistance vessel-sized arteries of BK channel deficient mice

Functional Roles of Ca v 1.3 (α 1D ) Calcium Channel in Sinoatrial Nodes

We directly examined the role of the Ca v 1.3 (α 1D ) Ca 2+ channel in the sinoatrial (SA) node by using Ca v 1.3 Ca 2+ channel-deficient mice. A previous report has shown that the null mutant (Ca v 1.3 −/− ) mice have sinus bradycardia with a prolonged PR interval. In the present study, we show that spontaneous action potentials recorded from the SA nodes show a significant decrease in the beating frequency and rate of diastolic depolarization in Ca v 1.3 −/− mice compared with their heterozygous (Ca v 1.3 +/− ) or wild-type (WT, Ca v 1.3 +/+ ) littermates, suggesting that the deficit is intrinsic to the SA node. Whole-cell L-type Ca 2+ currents ( I Ca,L s) recorded in single isolated SA node cells from Ca v 1.3 −/− mice show a significant depolarization shift in the activation threshold. The voltage-dependent activation of Ca v 1.2 (α 1C ) versus Ca v 1.3 Ca 2+ channel subunits was directly compared by using a heterologous expression system without β coexpression. Similar to the I Ca,L recorded in the SA node of Ca v 1.3 −/− mutant mice, the Ca v 1.2 Ca 2+ channel shows a depolarization shift in the voltage-dependent activation compared with that in the Ca v 1.3 Ca 2+ channel. In summary, using gene-targeted deletion of the Ca v 1.3 Ca 2+ channel, we were able to establish a role for Ca v 1.3 Ca 2+ channels in the generation of the spontaneous action potential in SA node cells.

A K(ATP) channel deficiency affects resting tension, not contractile force, during fatigue in skeletal muscle.

The objective of this study was to determine how an ATP-sensitive K(+) (K(ATP)) channel deficiency affects the contractile and fatigue characteristics of extensor digitorum longus (EDL) and soleus muscle of 2- to 3-mo-old and 1-yr-old mice. K(ATP) channel-deficient mice were obtained by disrupting the Kir6.2 gene that encodes for the protein forming the pore of the channel. At 2-3 mo of age, the force-frequency curve, the twitch, and the tetanic force of EDL and soleus muscle of K(ATP) channel-deficient mice were not significantly different from those in wild-type mice. However, the tetanic force and maximum rate of force development decreased with aging to a greater extent in EDL and soleus muscle of K(ATP) channel-deficient mice (24-40%) than in muscle of wild-type mice (7-17%). During fatigue, the K(ATP) channel deficiency had no effect on the decrease in tetanic force in EDL and soleus muscle, whereas it caused a significantly greater increase in resting tension when compared with muscle of wild-type mice. The recovery of tetanic force after fatigue was not affected by the deficiency in 2- to 3-mo-old mice, whereas in 1-yr-old mice, force recovery was significantly less in muscle of K(ATP) channel-deficient than wild-type mice. It is suggested that the major function of the K(ATP) channel during fatigue is to reduce the development of a resting tension and not to contribute to the decrease in force. It is also suggested that the K(ATP) channel plays an important role in protecting muscle function in older mice.

Immunohistochemical Localization of Calbindin-D28k in the Pancreas of Normal and KATP Channel-deficient Mice: Its Possible Role in the Prevention of Cell Damage to Islet Cells

Immunohistochemical Localization of Calbindin-D28k in the Pancreas of Normal and KATP Channel-deficient Mice: Its Possible Role in the Prevention of Cell Damage to Islet Cells

Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice.

ATP-sensitive K+ (KATP) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated KATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K+ ion-selective pore of the channel. The homozygous mice (Kir6.2(-/-)) lack KATP channel activity. Although the resting membrane potential and basal intracellular calcium concentrations ([Ca2+]i) of pancreatic beta cells in Kir6.2(-/-) are significantly higher than those in control mice (Kir6.2(+/+)), neither glucose at high concentrations nor the sulfonylurea tolbutamide elicits a rise in [Ca2+]i, and no significant insulin secretion in response to either glucose or tolbutamide is found in Kir6.2(-/-), as assessed by perifusion and batch incubation of pancreatic islets. Despite the defect in glucose-induced insulin secretion, Kir6.2(-/-) show only mild impairment in glucose tolerance. The glucose-lowering effect of insulin, as assessed by an insulin tolerance test, is increased significantly in Kir6.2(-/-), which could protect Kir6.2(-/-) from developing hyperglycemia. Our data indicate that the KATP channel in pancreatic beta cells is a key regulator of both glucose- and sulfonylurea-induced insulin secretion and suggest also that the KATP channel in skeletal muscle might be involved in insulin action.

Pleiotropic effects of a disrupted K+ channel gene: reduced body weight, impaired motor skill and muscle contraction, but no seizures.

To investigate the roles of K+ channels in the regulation and fine-tuning of cellular excitability, we generated a mutant mouse carrying a disrupted gene for the fast activating, voltage-gated K+ channel Kv3.1. Kv3.1-/- mice are viable and fertile but have significantly reduced body weights compared with their Kv3.1+/- littermates. Wild-type, heterozygous, and homozygous Kv3.1 channel-deficient mice exhibit similar spontaneous locomotor and exploratory activity. In a test for coordinated motor skill, however, homozygous Kv3.1-/- mice perform significantly worse than their heterozygous Kv3.1+/- or wild-type littermates. Both fast and slow skeletal muscles of Kv3.1-/- mice are slower to reach peak force and to relax after contraction, consequently leading to tetanic responses at lower stimulation frequencies. Both mutant muscles generate significantly smaller contractile forces during a single twitch and during tetanic conditions. Although Kv3.1-/- mutants exhibit a normal auditory frequency range, they show significant differences in their acoustic startle responses. Contrary to expectation, homozygous Kv3.1-/- mice do not have increased spontaneous seizure activity.