Vikram A. Kanda

Mind the Gap: Disparity Between Research Funding and Costs of Care for Diabetic Foot Ulcers

Diabetic foot ulceration (DFU) is a serious and prevalent complication of diabetes, ultimately affecting some 25% of those living with the disease (1). DFUs have a consistently negative impact on quality of life and productivity as diabetic patients report stigma, social isolation, unemployment, and depression (2–5). Patients with DFUs also have morbidity and mortality rates equivalent to aggressive forms of cancer (2). These ulcers remain an important risk factor for lower-extremity amputation as up to 85% of amputations are preceded by foot ulcers (6). It should therefore come as no surprise that some 33% of the

KCNQ1, KCNE2, and Na(+)-coupled solute transporters form reciprocally regulating complexes that affect neuronal excitability

116 billion in direct costs generated by the treatment of diabetes and its complications was linked to the treatment of foot ulcers (7). Another study has suggested that 25–50% of the costs related to inpatient diabetes care may be directly related to DFUs (2). National standards have been developed for DFU prevention and care (8–10). Given the high prevalence, severity, costs, and morbidity of diabetic foot complications, one would expect that federal funding for DFU research would be proportionate to its public health impact. The National Institutes of Health (NIH) is the major source of federal funding for medical research in the U.S. We therefore examined NIH funding for both diabetes and DFUs using the NIH Research Portfolio Online Reporting Tools (RePORT) from 2002 to 2011 (11). We also examined differences in the number of peer-reviewed publications (using PubMed at www.ncbi.nlm.nih.gov) on both diabetes and diabetic foot ulcers between the years 1980–2010. The search terms …

MinK-dependent internalization of the IKs potassium channel

Complexes of solute transporters and potassium channels that reciprocally regulate each other may contribute to seizure susceptibility. Stopping Seizures The activity of potassium channels limits neuronal excitability, and mutations in the regulatory subunit (KCNE2), which promotes the activity of the potassium-conducting pore (KCNQ1), are associated with increased seizure susceptibility. Abbott et al. found that SMIT1, which transports the molecule myo-inositol, associated with KCNQ1 or KCNQ1-KCNE2 complexes. When complexed with KCNE2, KCNQ1 is constitutively active. SMIT1 activity was increased in the presence of KCNQ1 but was inhibited in the presence of KCNQ1-KCNE2. SMIT1 increased the activity of both KCNQ1 and KCNQ1-KCNE2 complexes. The increased seizure activity of mice deficient in KCNE2 was attenuated by administration of myo-inositol, suggesting that a decrease in SMIT1 activity or alterations in the activity of these molecular complexes may contribute to seizure susceptibility. Na+-coupled solute transport is crucial for the uptake of nutrients and metabolic precursors, such as myo-inositol, an important osmolyte and precursor for various cell signaling molecules. We found that various solute transporters and potassium channel subunits formed complexes and reciprocally regulated each other in vitro and in vivo. Global metabolite profiling revealed that mice lacking KCNE2, a K+ channel β subunit, showed a reduction in myo-inositol concentration in cerebrospinal fluid (CSF) but not in serum. Increased behavioral responsiveness to stress and seizure susceptibility in Kcne2−/− mice were alleviated by injections of myo-inositol. Suspecting a defect in myo-inositol transport, we found that KCNE2 and KCNQ1, a voltage-gated potassium channel α subunit, colocalized and coimmunoprecipitated with SMIT1, a Na+-coupled myo-inositol transporter, in the choroid plexus epithelium. Heterologous coexpression demonstrated that myo-inositol transport by SMIT1 was augmented by coexpression of KCNQ1 but was inhibited by coexpression of both KCNQ1 and KCNE2, which form a constitutively active, heteromeric K+ channel. SMIT1 and the related transporter SMIT2 were also inhibited by a constitutively active mutant form of KCNQ1. The activities of KCNQ1 and KCNQ1-KCNE2 were augmented by SMIT1 and the glucose transporter SGLT1 but were suppressed by SMIT2. Channel-transporter signaling complexes may be a widespread mechanism to facilitate solute transport and electrochemical crosstalk.

KCNE2 forms potassium channels with KCNA3 and KCNQ1 in the choroid plexus epithelium

AIMS KCNQ1-MinK potassium channel complexes (4alpha:2beta stoichiometry) generate IKs, the slowly activating human cardiac ventricular repolarization current. The MinK ancillary subunit slows KCNQ1 activation, eliminates its inactivation, and increases its unitary conductance. However, KCNQ1 transcripts outnumber MinK transcripts five to one in human ventricles, suggesting KCNQ1 also forms other heteromeric or even homomeric channels there. Mechanisms governing which channel types prevail have not previously been reported, despite their significance: normal cardiac rhythm requires tight control of IKs density and kinetics, and inherited mutations in KCNQ1 and MinK can cause ventricular fibrillation and sudden death. Here, we describe a novel mechanism for this control. METHODS AND RESULTS Whole-cell patch-clamping, confocal immunofluorescence microscopy, antibody feeding, biotin feeding, fluorescent transferrin feeding, and protein biochemistry techniques were applied to COS-7 cells heterologously expressing KCNQ1 with wild-type or mutant MinK and dynamin 2 and to native IKs channels in guinea-pig myocytes. KCNQ1-MinK complexes, but not homomeric KCNQ1 channels, were found to undergo clathrin- and dynamin 2-dependent internalization (DDI). Three sites on the MinK intracellular C-terminus were, in concert, necessary and sufficient for DDI. Gating kinetics and sensitivity to XE991 indicated that DDI decreased cell-surface KCNQ1-MinK channels relative to homomeric KCNQ1, decreasing whole-cell current but increasing net activation rate; inhibiting DDI did the reverse. CONCLUSION The data redefine MinK as an endocytic chaperone for KCNQ1 and present a dynamic mechanism for controlling net surface Kv channel subunit composition-and thus current density and gating kinetics-that may also apply to other alpha-beta type Kv channel complexes.

Protein kinase C downregulates IKs by stimulating KCNQ1-KCNE1 potassium channel endocytosis

Cerebrospinal fluid (CSF) is crucial for normal function and mechanical protection of the CNS. The choroid plexus epithelium (CPe) is primarily responsible for secreting CSF and regulating its composition by mechanisms currently not fully understood. Previously, the heteromeric KCNQ1‐KCNE2 K+ channel was functionally linked to epithelial processes including gastric acid secretion and thyroid hormone biosynthesis. Here, using Kcne2–/– tissue as a negative control, we found cerebral expression of KCNE2 to be markedly enriched in the CPe apical membrane, where we also discovered expression of KCNQ1. Targeted Kcne2 gene deletion in C57B6 mice increased CPe outward K+ current 2‐fold. The Kcne2 deletion‐enhanced portion of the current was inhibited by XE991 (10 μM) and margatoxin (10 μM) but not by dendrotoxin (100 nM), indicating that it arose from augmentation of KCNQ subfamily and KCNA3 but not KCNA1 K+ channel activity. Kcne2 deletion in C57B6 mice also altered the polarity of CPe KCNQ1 and KCNA3 trafficking, hyperpolarized the CPe membrane by 9 ± 2 mV, and increased CSF [Cl–] by 14% compared with wild‐type mice. These findings constitute the first report of CPe dysfunction caused by cation channel gene disruption and suggest that KCNE2 influences blood‐CSF anion flux by regulating KCNQ1 and KCNA3 in the CPe.—Roepke, T. K., Kanda, V. A., Purtell, K., King, E. C., Lerner, D. J., Abbott, G. W. KCNE2 forms potassium channels with KCNA3 and KCNQ1 in the choroid plexus epithelium. FASEB J. 25, 4264–4273 (2011). www.fasebj.org

Genetic dissection reveals unexpected influence of beta subunits on KCNQ1 K+ channel polarized trafficking in vivo

BACKGROUND The slow-activating cardiac repolarization K(+) current (I(Ks)), generated by the KCNQ1-KCNE1 potassium channel complex, is controlled via sympathetic and parasympathetic regulation in vivo. Inherited KCNQ1 and KCNE1 mutations predispose to ventricular fibrillation and sudden death, often triggered by exercise or emotional stress. Protein kinase C (PKC), which is activated by α1 adrenergic receptor stimulation, is known to downregulate I(Ks) via phosphorylation of KCNE1 serine 102, but the underlying mechanism has remained enigmatic. We previously showed that KCNE1 mediates dynamin-dependent endocytosis of KCNQ1-KCNE1 complexes. OBJECTIVE This study sought to determine the potential role of endocytosis in I(Ks) downregulation by PKC. METHODS We utilized patch clamping and fluorescence microscopy to study Chinese hamster ovary (CHO) cells coexpressing KCNQ1, KCNE1, and wild-type or dominant-negative mutant (K44A) dynamin 2, and neonatal mouse ventricular myocytes. RESULTS The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased I(Ks) density by >60% (P < .05) when coexpressed with wild-type dynamin 2 in CHO cells, but had no effect when coexpressed with K44A-dynamin 2. Thus, functional dynamin was required for downregulation of I(Ks) by PKC activation. PMA increased KCNQ1-KCNE1 endocytosis in CHO cells expressing wild-type dynamin 2, but had no effect on KCNQ1-KCNE1 endocytosis in CHO cells expressing K44A-dynamin 2, determined using the Pearson correlation coefficient to quantify endosomal colocalization of KCNQ1 and KCNE1 with internalized fluorescent transferrin. KCNE1-S102A abolished the effect of PMA on I(Ks) currents and endocytosis. Importantly, PMA similarly stimulated endocytosis of endogenous KCNQ1 and KCNE1 in neonatal mouse myocytes. CONCLUSION PKC activation downregulates I(Ks) by stimulating KCNQ1-KCNE1 channel endocytosis.

KCNE Regulation of K+ Channel Trafficking – a Sisyphean Task?

Targeted deletion of the Kcne2 potassium channel β subunit gene ablates gastric acid secretion and predisposes to gastric neoplasia in mice. Here, we discovered that Kcne2 deletion basolaterally reroutes the Kcnq1 α subunit in vivo in parietal cells (PCs), in which the normally apical location of the Kcnq1‐Kcne2 channel facilitates its essential role in gastric acid secretion. Quantitative RT‐PCR and Western blotting revealed that Kcne2 deletion remodeled fundic Kcne3 (2.9±0.8‐fold mRNA increase, n=10;5.3± 0.4‐fold protein increase, n=7) but not Kcne1, 4, or 5, and resulted in basolateral Kcnq1‐Kcne3 complex formation in Kcne2−/− PCs. Concomitant targeted deletion of Kcne3 (creating Kcne2−/−Kcne3−/− mice) restored PC apical Kcnq1 localization without Kcnel, 4, or 5 remodeling (assessed by quantitative RT‐PCR;n=5–10), indicating Kcne3 actively, basolaterally rerouted Kcnq1 in Kcne2−/− PCs. Despite this, Kcne3 deletion exacerbated gastric hyperplasia in Kcne2−/− mice, and both hypochlo‐rhydria and hyperplasia in Kcne2+/− mice, suggesting that Kcne3 up‐regulation was beneficial in Kcne2‐depleted PCs. The findings reveal, in vivo, Kcne‐dependent α subunit polarized trafficking and the existence and consequences of potassium channel β subunit remodeling.—Roepke, T. K., King, E. C., Purtell, K., Kanda, V. A., Lerner, D. J., Abbott, G. W. Genetic dissection reveals unexpected influence of β subunits on KCNQ1 K+ channel polarized trafficking in vivo. FASEB J. 25, 727–736 (2011). www.fasebj.org

KCNE1 and KCNE2 Provide a Checkpoint Governing Voltage-Gated Potassium Channel α-Subunit Composition

Voltage-gated potassium (Kv) channels shape the action potentials of excitable cells and regulate membrane potential and ion homeostasis in excitable and non-excitable cells. With 40 known members in the human genome and a variety of homomeric and heteromeric pore-forming α subunit interactions, post-translational modifications, cellular locations, and expression patterns, the functional repertoire of the Kv α subunit family is monumental. This versatility is amplified by a host of interacting proteins, including the single membrane-spanning KCNE ancillary subunits. Here, examining both the secretory and the endocytic pathways, we review recent findings illustrating the surprising virtuosity of the KCNE proteins in orchestrating not just the function, but also the composition, diaspora and retrieval of channels formed by their Kv α subunit partners.

Targeted deletion of Kcne2 impairs HCN channel function in mouse thalamocortical circuits.

Voltage-gated potassium (Kv) currents generated by N-type α-subunit homotetramers inactivate rapidly because an N-terminal ball domain blocks the channel pore after activation. Hence, the inactivation rate of heterotetrameric channels comprising both N-type and non-N-type (delayed rectifier) α-subunits depends upon the number of N-type α-subunits in the complex. As Kv channel inactivation and inactivation recovery rates regulate cellular excitability, the composition and expression of these heterotetrameric complexes are expected to be tightly regulated. In a companion article, we showed that the single transmembrane segment ancillary (β) subunits KCNE1 and KCNE2 suppress currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels, by trapping them early in the secretory pathway. Here, we show that this trapping is prevented by coassembly of the N-type α-subunits with intra-subfamily delayed rectifier α-subunits. Extra-subfamily delayed rectifier α-subunits, regardless of their capacity to interact with KCNE1 and KCNE2, cannot rescue Kv1.4 or Kv3.4 surface expression unless engineered to interact with them using N-terminal A and B domain swapping. The KCNE1/2-enforced checkpoint ensures N-type α-subunits only reach the cell surface as part of intra-subfamily mixed-α complexes, thereby governing channel composition, inactivation rate, and-by extension-cellular excitability.

Transcriptomic analysis reveals atrial KCNE1 down‐regulation following lung lobectomy

Background Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the pacemaking current, Ih, which regulates neuronal excitability, burst firing activity, rhythmogenesis, and synaptic integration. The physiological consequence of HCN activation depends on regulation of channel gating by endogenous modulators and stabilization of the channel complex formed by principal and ancillary subunits. KCNE2 is a voltage-gated potassium channel ancillary subunit that also regulates heterologously expressed HCN channels; whether KCNE2 regulates neuronal HCN channel function is unknown. Methodology/Principal Findings We investigated the effects of Kcne2 gene deletion on Ih properties and excitability in ventrobasal (VB) and cortical layer 6 pyramidal neurons using brain slices prepared from Kcne2 +/+ and Kcne2 −/− mice. Kcne2 deletion shifted the voltage-dependence of Ih activation to more hyperpolarized potentials, slowed gating kinetics, and decreased Ih density. Kcne2 deletion was associated with a reduction in whole-brain expression of both HCN1 and HCN2 (but not HCN4), although co-immunoprecipitation from whole-brain lysates failed to detect interaction of KCNE2 with HCN1 or 2. Kcne2 deletion also increased input resistance and temporal summation of subthreshold voltage responses; this increased intrinsic excitability enhanced burst firing in response to 4-aminopyridine. Burst duration increased in corticothalamic, but not thalamocortical, neurons, suggesting enhanced cortical excitatory input to the thalamus; such augmented excitability did not result from changes in glutamate release machinery since miniature EPSC frequency was unaltered in Kcne2 −/− neurons. Conclusions/Significance Loss of KCNE2 leads to downregulation of HCN channel function associated with increased excitability in neurons in the cortico-thalamo-cortical loop. Such findings further our understanding of the normal physiology of brain circuitry critically involved in cognition and have implications for our understanding of various disorders of consciousness.