Sangita P. Patel

Transient outward potassium current, 'Ito', phenotypes in the mammalian left ventricle: underlying molecular, cellular and biophysical mechanisms.

At least two functionally distinct transient outward K+ current (Ito) phenotypes can exist across the free wall of the left ventricle (LV). Based upon their voltage‐dependent kinetics of recovery from inactivation, these two phenotypes are designated ‘Ito,fast’ (recovery time constants on the order of tens of milliseconds) and ‘Ito,slow’ (recovery time constants on the order of thousands of milliseconds). Depending upon species, either Ito,fast, Ito,slow or both current phenotypes may be expressed in the LV free wall. The expression gradients of these two Ito phenotypes across the LV free wall are typically heterogeneous and, depending upon species, may consist of functional phenotypic gradients of both Ito,fast and Ito,slow and/or density gradients of either phenotype. We review the present evidence (molecular, biophysical, electrophysiological and pharmacological) for Kv4.2/4.3 α subunits underlying LV Ito,fast and Kv1.4 α subunits underlying LV Ito,slow and speculate upon the potential roles of each of these currents in determining frequency‐dependent action potential characteristics of LV subepicardial versus subendocardial myocytes in different species. We also review the possible functional implications of (i) ancillary subunits that regulate Kv1.4 and Kv4.2/4.3 (Kvβ subunits, DPPs), (ii) KChIP2 isoforms, (iii) spider toxin‐mediated block of Kv4.2/4.3 (Heteropoda toxins, phrixotoxins), and (iv) potential mechanisms of modulation of Ito,fast and Ito,slow by cellular redox state, [Ca2+]i and kinase‐mediated phosphorylation. Ito phenotypic activation and state‐dependent gating models and molecular structure–function relationships are also discussed.

Heterogeneous expression of KChIP2 isoforms in the ferret heart

Kv4 channels are believed to underlie the rapidly recovering cardiac transient outward current (Ito) phenotype. However, heterologously expressed Kv4 channels fail to fully reconstitute the native current. Kv channel interacting proteins (KChIPs) have been shown to modulate Kv4 channel function. To determine the potential involvement of KChIPs in the rapidly recovering Ito, we cloned three KChIP2 isoforms (designated fKChIP2, 2a and 2b) from the ferret heart. Based upon immunoblot data suggesting the presence of a potential endogenous KChIP‐like protein in HEK 293, CHO and COS cells but absence in Xenopus oocytes, we coexpressed Kv4.3 and the fKChIP2 isoforms in Xenopus oocytes. Functional analysis showed that while all fKChIP2 isoforms produced a fourfold acceleration of recovery kinetics compared to Kv4.3 expressed alone, only fKChIP2a produced large depolarizing shifts in the V1/2 of steady‐state activation and inactivation as seen for the native rapidly recovering Ito. Analysis of RNA and protein expression of the three fKChIP2 isoforms in ferret ventricles showed that fKChIP2b was most abundant and was expressed in a gradient paralleling the rapidly recovering Ito distribution. Ferret KChIP2 and 2a were expressed at very low levels. The ventricular expression distribution suggests that fKChIP2 isoforms are involved in modulation of the rapidly recovering Ito; however, additional regulatory factors are also likely to be involved in generating the native current.

Elucidating KChIP effects on Kv4.3 inactivation and recovery kinetics with a minimal KChIP2 isoform.

Kv channel interacting proteins (KChIPs) are Ca2+‐binding proteins with four EF‐hands. KChIPs modulate Kv4 channel gating by slowing inactivation kinetics and accelerating recovery kinetics. Thus, KChIPs are believed to be important regulators of Kv4 channels underlying transient outward K+ currents in many excitable cell types. We have cloned a structurally minimal KChIP2 isoform (KChIP2d) from ferret heart. KChIP2d corresponds to the final 70 C‐terminal amino acids of other KChIPs and has only one EF‐hand. We demonstrate that KChIP2d is a functional KChIP that both accelerates recovery and slows inactivation kinetics of Kv4.3, indicating that the minimal C‐terminus can maintain KChIP regulatory properties. We utilize KChIP2d to further demonstrate that: (i) the EF‐hand modulates effects on Kv4.3 inactivation but not recovery; (ii) Ca2+‐dependent effects on Kv4.3 inactivation are mediated through a mechanism reflected in the slow time constant of inactivation; and (iii) a short stretch of amino acids exclusive of the EF‐hand partially mediates Ca2+‐independent effects on recovery. Our results demonstrate that distinct regions of a KChIP molecule are involved in modulating inactivation and recovery. The potential ability of KChIP EF‐hands to sense intracellular Ca2+ levels and transduce these changes to alterations in Kv4 channel inactivation kinetics may serve as a mechanism allowing intracellular Ca2+ transients to modulate repolarization. KChIP2d is a valuable tool for elucidating structural domains of KChIPs involved in Kv4 channel regulation.

Regulation of Kv4.3 voltage-dependent gating kinetics by KChIP2 isoforms

We conducted a kinetic analysis of the voltage dependence of macroscopic inactivation (τfast, τslow), closed‐state inactivation (τclosed,inact), recovery (τrec), activation (τact), and deactivation (τdeact) of Kv4.3 channels expressed alone in Xenopus oocytes and in the presence of the calcium‐binding ancillary subunits KChIP2b and KChIP2d. We demonstrate that for all expression conditions, τrec, τclosed,inact and τfast are components of closed‐state inactivation transitions. The values of τclosed,inact and τfast monotonically merge from −30 to −20 mV while the values of τclosed,inact and τrec approach each other from −60 to −50 mV. These data generate classic bell‐shaped time‐constant–potential curves. With the KChIPs, these curves are distinct from that of Kv4.3 expressed alone due to acceleration of τrec and slowing of τclosed,inact and τfast. Only at depolarized potentials where channels open is τslow detectable suggesting that it represents an open‐state inactivation mechanism. With increasing depolarization, KChIPs favour this open‐state inactivation mechanism, supported by the observation of larger transient reopening currents upon membrane hyperpolarization compared to Kv4.3 expressed alone. We propose a Kv4.3 gating model wherein KChIP2 isoforms accelerate recovery, slow closed‐state inactivation, and promote open‐state inactivation. This model supports the observations that with KChIPs, closed‐state inactivation transitions are [Ca2+]i‐independent, while open‐state inactivation is [Ca2+]i‐dependent. The selective KChIP‐ and Ca2+‐dependent modulation of Kv4.3 inactivation mechanisms predicted by this model provides a basis for dynamic modulation of the native cardiac transient outward current by intracellular Ca2+ fluxes during the action potential.

Kinetic properties of Kv4.3 and their modulation by KChIP2b.

KChIPs are a family of Kv4 K(+) channel ancillary subunits whose effects usually include slowing of inactivation, speeding of recovery from inactivation, and increasing channel surface expression. We compared the effects of the 270 amino acid KChIP2b on Kv4.3 and a Kv4.3 inner pore mutant [V(399, 401)I]. Kv4.3 showed fast inactivation with a bi-exponential time course in which the fast time constant predominated. KChIP2b expressed with wild-type Kv4.3 slowed the fast time constant of inactivation; however, the overall rate of inactivation was faster due to reduction of the contribution of the slow inactivation phase. Introduction of [V(399, 401)I] slowed both time constants of inactivation less than 2-fold. Inactivation was incomplete after 20s pulse durations. Co-expression of KChIP2b with Kv4.3 [V(399, 401)I] slowed inactivation dramatically. KChIP2b increased the rate of recovery from inactivation 7.6-fold in the wild-type channel and 5.7-fold in Kv4.3 [V(399,401)I]. These data suggest that inner pore structure is an important factor in the modulatory effects of KChIP2b on Kv4.3 K(+) channels.

Corneal Lymphangiogenesis: Implications in Immunity

The lymphatic system is a network of vessels throughout the body that serves to return lymphatic fluid to the systemic blood circulation and serves as the immune systems highway in the afferent limb of the immune response. This paper reviews the basics of structure, function and development of the lymphatic system. The emphasis is on understanding the role of lymphatics in the cornea as related to angiogenesis and the clinical setting of corneal transplant rejection. The lymphatic system has two main functions: to drain excess extracellular fluid from capillary beds to return to the systemic blood circulation and to capture antigen for presentation to the immune system in the lymphoid compartment (e.g. nodes) connected in parallel to the lymphatic system. The normal cornea is devoid of lymphatic vessels and blood vessels, thus allowing for its unique immune privileged status. Abnormal blood vessel growth into the cornea has been studied for many years, but the study of lymphatic vessels has been hampered until the recent discovery of lymphatic-specific markers. Subsequent studies of vascularized corneas support the presence of lymphatic vessels in the cornea. The growth of new lymphatic (lymphangiogenesis) and blood (hemangiogenesis) vessels in the cornea is closely related to the abrogation of the immune privileged status of the cornea. This review summarizes our current understanding of the parallel development of hemangiogenesis and lymphangiogenesis as related to the mechanisms of immune rejection of corneal transplants.

Corneal Endothelial Cell Proliferation: A Function of Cell Density

PURPOSE To determine how stimuli that increase corneal endothelial cell proliferation of human corneas in culture relate to changes in endothelial cell density in the central and peripheral cornea. METHODS Human donor cadaver corneas not suitable for transplantation were divided into four pie-shaped wedges and incubated at 37 degrees C in medium supplemented with fetal bovine serum, epidermal growth factor, fibroblast growth factor, and gentamicin. To promote a proliferative response, samples were treated with EDTA at concentrations of 0, 0.5, 2.5, and 5.0 mM for 1 hour and then returned to culture medium. Endothelial cell proliferation was assayed with Ki-67 immunolocalization 48 and 96 hours after EDTA treatment. Samples were mounted with propidium iodide or DAPI. The total number of cells and the number of Ki-67-positive cells were counted in three regions, defined as central, mid, and peripheral cornea, to determine endothelial cell density and percentage of proliferation. RESULTS A proliferative response to EDTA was not found. However, increased proliferation was noted in the central compared with the peripheral corneal region. Unexpectedly, the increased proliferation in the central region corresponded to a trend of lower endothelial cell density in the central region compared with the peripheral region. Corneal endothelial cell proliferation under our culture conditions is noted primarily when cell density is less than 2000 cells/mm(2). CONCLUSIONS Corneal endothelial cell proliferation under our culture conditions does not lead to supranormal endothelial cell density. Rather, cell proliferation is noted in those regions that may be experiencing a greater burden of cell loss.

SLC4A11 and the Pathophysiology of Congenital Hereditary Endothelial Dystrophy

Congenital hereditary endothelial dystrophy (CHED) is a rare autosomal recessive disorder of the corneal endothelium characterized by nonprogressive bilateral corneal edema and opacification present at birth. Here we review the current knowledge on the role of the SLC4A11 gene, protein, and its mutations in the pathophysiology and clinical presentation of CHED. Individuals with CHED have mutations in SLC4A11 which encodes a transmembrane protein in the SLC4 family of bicarbonate transporters. The expression of SLC4A11 in the corneal endothelium and inner ear patterns the deficits seen in CHED with corneal edema and hearing loss (Harboyan syndrome). slc4a11-null-mouse models recapitulate the CHED disease phenotype, thus establishing a functional role for SLC4A11 in CHED. However, the transport function of SLC4A11 remains unsettled. Some of the roles that have been attributed to SLC4A11 include H+ and NH4 + permeation, electrogenic Na+-H+ exchange, and water transport. Future studies of the consequences of SLC4A11 dysfunction as well as further understanding of corneal endothelial ion transport will help clarify the involvement of SLC4A11 in the pathophysiology of CHED.

A practice model for trabecular meshwork surgery.

Models for practicing ophthalmic surgery are a necessary part of all ophthalmic training. Herein, we describe a method for preparing human donor cadaveric eyes with direct visualization of the anterior chamber angle for practicing trabecular meshwork surgery. This model can be adapted for use with both fresh and formalin-fixed eyes. The use of formalin-fixed eyes decreases the risk of infection transmission inherent with human cadaveric eyes, allows prepared eyes to be stored indefinitely, and can be reused until all of the trabecular meshwork has been exhausted in surgery. We describe the application of this model for the technique of ab interno trabeculectomy using the Trabectome system for treating open-angle glaucoma.

Carbonic anhydrase inhibitors in corneal endothelial transport.

PURPOSE Carbonic anhydrases play a central buffering role in current models of fluid transport in corneal endothelium, but in humans, clinical use of carbonic anhydrase inhibitors (CAIs) for the management of glaucoma does not cause corneal swelling. This study compares species differences in response to CAIs in human versus bovine corneal endothelial transport. METHODS Short-circuit current (Isc) measurements were performed on bovine and human corneal endothelium under identical conditions. The effects of four CAIs (acetazolamide, brinzolamide, dorzolamide, and ethoxzolamide) were measured. Endothelial expression of carbonic anhydrase II and IV was evaluated by immunofluorescence microscopy. Functional presence of carbonic anhydrase activity was determined using the Hanssons cobalt sulfide histochemical method. RESULTS All four CAIs decreased bovine Isc (% change in Isc: acetazolamide, -21.0 ± 9.5, n = 8; brinzolamide, -35.5 ± 13.5, n = 9; dorzolamide, -33.6 ± 7.2, n = 8; ethoxzolamide, -35.3 ± 12.9, n = 8). That decrease was not present in humans (% change in Isc: acetazolamide, 16.2 ± 20.1, n = 3; brinzolamide, 6.7 ± 13.9, n = 3; dorzolamide, 8.0 ± 20.4, n = 3; ethoxzolamide, -4.8 ± 10.3, n = 2). Despite no functional effect of CAIs on Isc, both carbonic anhydrase II and IV were present in human corneal endothelium by immunofluorescence microscopy. Histochemical analysis of human corneal endothelium revealed functionally active carbonic anhydrase activity inhibited by brinzolamide. CONCLUSIONS Carbonic anhydrase facilitates ion transport impacting the corneal endothelial Isc in bovine but not human corneal endothelium, despite its presence and functional activity in human tissue. This finding supports the clinical observation of no corneal swelling in humans administered CAIs and suggests that alternative ion transport mechanisms may be operational in corneal endothelium of different species.