Faculty Bibliography

Cardiac KATP channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice which express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression with the alpha-myosin heavy chain promoter. Weight gain and development after birth of these mice were similar to wild-type mice, but an increased mortality was noted after the age of 4-5 months. Transgenic mice lacked cardiac KATP channel activity as assessed with patch clamp techniques. Consistent with a decreased current density observed at positive voltages, the action potential duration was increased in these mice. Some myocytes developed early afterdepolarizations following isoproterenol treatment. Hemodynamic measurements revealed no significant effects on ventricular function (apart from a slightly elevated heart rate) whereas in-vivo electrophysiological recordings revealed a prolonged ventricular effective refractory period in transgenic mice. The transgenic mice tolerated stress less well as evident from treadmill stress tests. The pro-arrhythmogenic features and lack of adaptation to a stress response in transgenic mice suggests that these features are intrinsic to the myocardium and that KATP channels in the myocardium have an important role in protecting the heart from lethal arrhythmias and adaptation to stress situations

Intracellular Ca2+ levels rapidly rise following cross-linking of the T-cell receptor (TCR) and function as a critical intracellular second messenger in T-cell activation. It has been relatively under appreciated that K+ channels play an important role in Ca2+ influx into T lymphocytes by helping to maintain a negative membrane potential which provides an electrochemical gradient to drive Ca2+ influx. Here we show that the Ca2+-activated K+ channel, KCa3.1, which is critical for Ca2+ influx in reactivated naive T cells and central memory T cells, requires phosphatidylinositol-3 phosphatase [PI(3)P] for activation and is inhibited by the PI(3)P phosphatase myotubularin-related protein 6 (MTMR6). Moreover, by inhibiting KCa3.1, MTMR6 functions as a negative regulator of Ca2+ influx and proliferation of reactivated human CD4 T cells. These findings point to a new and unexpected role for PI(3)P and the PI(3)P phosphatase MTMR6 in the regulation of Ca2+ influx in activated CD4 T cells and suggest that MTMR6 plays a critical role in setting a minimum threshold for a stimulus to activate a T cell

Monitoring Editor: Guido Guidotti KCa3.1 is an intermediate conductance Ca(2+)-activated K channels that is expressed predominantly in hematopoietic cells, smooth muscle cells, and epithelia where it functions to regulate membrane potential, Ca2+ influx, cell volume and chloride secretion. We recently found that the KCa3.1 channel also specifically requires PI(3)P for channel activity and is inhibited by myotubularin related protein 6 (MTMR6), a PI(3)P phosphatase. We now show that PI(3)P indirectly activates KCa3.1. Unlike KCa3.1 channels, the related KCa2.1, KCa2.2 or KCa2.3 channels do not require PI(3)P for activity, suggesting that the KCa3.1 channel has evolved a unique means of regulation that is critical for their biological function. By making chimeric channels between KCa3.1 and KCa2.3, we identified a stretch of 14 amino acids in the carboxyterminal calmodulin binding domain of KCa3.1 that is sufficient to confer regulation of KCa2.3 by PI(3)P. However, mutation of a single potential phosphorylation site in these 14 amino acids did not affect channel activity. These data when taken together suggest that PI(3)P and these 14 amino acids regulate KCa3.1 channel activity by recruiting an as yet to be defined regulatory subunit that is required for Ca2+ gating of KCa3.1

The regulation of ATP-sensitive potassium (K(ATP)) channel activity is complex and a multitude of factors determine their open probability. Physiologically and pathophysiologically, the most important of these are intracellular nucleotides, with a long-recognized role for glycolytically derived ATP in regulating channel activity. To identify novel regulatory subunits of the K(ATP) channel complex, we performed a two-hybrid protein-protein interaction screen, using as bait the mouse Kir6.2 C terminus. Screening a rat heart cDNA library, we identified two potential interacting proteins to be the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triose-phosphate isomerase. The veracity of interaction was verified by co-immunoprecipitation techniques in transfected mammalian cells. We additionally demonstrated that pyruvate kinase also interacts with Kir6.2 subunits. The physiological relevance of these interactions is illustrated by the demonstration that native Kir6.2 protein similarly interact with GAPDH and pyruvate kinase in rat heart membrane fractions and that Kir6.2 protein co-localize with these glycolytic enzymes in rat ventricular myocytes. The functional relevance of our findings is demonstrated by the ability of GAPDH or pyruvate kinase substrates to directly block the K(ATP) channel under patch clamp recording conditions. Taken together, our data provide direct evidence for the concept that key enzymes involved in glycolytic ATP production are part of a multisubunit K(ATP) channel protein complex. Our data are consistent with the concept that the activity of these enzymes (possibly by ATP formation in the immediate intracellular microenvironment of this macromolecular K(ATP) channel complex) causes channel closure

Prevailing data suggest that sarcolemmal ATP-sensitive (K(ATP)) channels in the adult heart consist of Kir6.2 and SUR2A subunits, but the expression of other K(ATP) channel subunits (including SUR1, SUR2B, and Kir6.1) is poorly defined. The situation is even less clear for the immature heart, which shows a remarkable resistance to hypoxia and metabolic stress. The hypoxia-induced action potential shortening and opening of sarcolemmal K(ATP) channels that occurs in adults is less prominent in the immature heart. This might be due in part to the different biophysical and pharmacological properties of K(ATP) channels of immature and adult K(ATP) channels. Because these properties are largely conferred by subunit composition, it is important to examine the relative expression levels of the various K(ATP) channel subunits during maturation. We therefore used RNAse protection assays, reverse transcription-PCR approaches, and Western blotting to characterize the mRNA and protein expression profiles of K(ATP) channel subunits in fetal, neonatal, and adult mouse heart. Our data indicate that each of the K(ATP) channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B) is expressed in the mouse heart at all of the developmental time points studied. However, the expression level of each of the subunits is low in the fetal heart and progressively increases with maturation. Each of the subunits seems to be expressed in ventricular myocytes with a subcellular expression pattern matching that found in the adult. Our data suggest that the K(ATP) channel composition may change during maturation, which has important implications for K(ATP) channel function in the developing heart

Myotubularins (MTMs) belong to a large subfamily of phosphatases that dephosphorylate the 3' position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P(2). MTM1 is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth syndrome. However, little is known about the general mechanism(s) whereby MTMs are regulated or the specific biological processes regulated by the different MTMs. We identified a Ca(2+)-activated K channel, K(Ca)3.1 (also known as KCa4, IKCa1, hIK1, or SK4), that specifically interacts with the MTMR6 subfamily of MTMs via coiled coil (CC) domains on both proteins. Overexpression of MTMR6 inhibited K(Ca)3.1 channel activity, and this inhibition required MTMR6's CC and phosphatase domains. This inhibition is specific; MTM1, a closely related MTM, did not inhibit K(Ca)3.1. However, a chimeric MTM1 in which the MTM1 CC domain was swapped for the MTMR6 CC domain inhibited K(Ca)3.1, indicating that MTM CC domains are sufficient to confer target specificity. K(Ca)3.1 was also inhibited by the PI(3) kinase inhibitors LY294002 and wortmannin, and this inhibition was rescued by the addition of PI(3)P, but not other phosphoinositides, to the patch pipette solution. PI(3)P also rescued the inhibition of K(Ca)3.1 by MTMR6 overexpression. These data, when taken together, indicate that K(Ca)3.1 is regulated by PI(3)P and that MTMR6 inhibits K(Ca)3.1 by dephosphorylating the 3' position of PI(3)P, possibly leading to decreased PI(3)P in lipid microdomains adjacent to K(Ca)3.1. K(Ca)3.1 plays important roles in controlling proliferation by T cells, vascular smooth muscle cells, and some cancer cell lines. Thus, our findings not only provide unique insights into the regulation of K(Ca)3.1 channel activity but also raise the possibility that MTMs play important roles in the negative regulation of T cells and in conditions associated with pathological cell proliferation, such as cancer and atherosclerosis

Contractions in neonatal rabbit ventricular myocytes seem to depend predominantly on Ca2+ influx through the Na+-Ca2+ exchanger (NCX). Unexpectedly, neonates are sensitive to the negative inotropic effect of L-type Ca2+ channel blockers. L-type Ca2+ channel blockers depress contractile function indirectly in neonatal myocytes by shortening the action potential duration (APD), thereby decreasing the influx of activator Ca2+ through the NCX. Freshly isolated ventricular myocytes from adult and neonatal (1-5 d) rabbits were electrically stimulated (0.5 Hz; 35 degrees C) while action potential (AP) and Ca2+ transients (Indo-1) were recorded in the absence and presence of nifedipine (10 microM). In separate experiments, cells were voltage-clamped with a constant AP waveform (APD90=170 ms) to determine the effect of nifedipine on Ca2+ transients independent from effects on the AP. Voltage-clamp experiments confirmed that nifedipine blocks L-type Ca2+ current in neonatal myocytes. Nifedipine markedly reduced Ca2+ transient amplitude and APD in both adults (transient=20 +/- 7%; APD90=31 +/- 4% of control) and neonates (transient=38 +/- 10%; APD90=57 +/- 6% of control). When the AP was held constant by voltage clamping, nifedipine significantly reduced the amplitude of Ca2+ transients in adults (27 +/- 9% of control) but had no effect on Ca2+ transient amplitude in neonatal myocytes. These results are consistent with the concept that immature ventricular myocytes are less reliant on L-type calcium channels and are more dependent on NCX for contraction. The negative inotropic effect of L-type Ca2+ channel blockers in neonates is attributable to shortening of the AP

The Na+-Ca2+ exchanger (NCX) is up-regulated in the neonatal rabbit heart. Because the duration of membrane depolarization is an important determinant of calcium entry via NCX, pharmacological agents that lengthen the action potential (AP) may significantly increase the amount of activator calcium in newborns. We tested this potentially novel therapeutic strategy by using action potential voltage clamp steps or using dofetilide, a blocker of IKr, to prolong the action potential duration (APD). The effects of changing APD on calcium transients were determined in ventricular myocytes at different developmental stages: newborn (1-4 days), juvenile (9-10 days), and adult ventricular myocytes (35 degrees C; 1 Hz). Calcium transient amplitude in neonatal myocytes increased substantially with clamping with longer APs. In contrast, exposure to dofetilide (0.1, 1, and 10 microM) under current clamp conditions increased APD in a concentration-dependent manner but had no significant effect on calcium transient amplitude in either neonates or adults. When the AP was held constant under voltage clamp conditions, dofetilide decreased the calcium transient amplitude in neonates. This effect is likely related to inhibition of sodium-calcium exchanger and L-type Ca2+ currents (ICa), as observed in separate experiments. These results suggest that dofetilide has a paradoxical effect on APD and calcium transients in the newborn heart