Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes

Abstract

Membrane lipid composition is a major determinant of cell excitability. In this study, we assessed the role of membrane cholesterol composition in the distribution and function of Kv1.5-based channels in rat cardiac membranes. In isolated rat atrial myocytes, the application of methyl-beta-cyclodextrin (MCD), an agent that depletes membrane cholesterol, caused a delayed increase in the Kv1.5-based sustained component, I(kur), which reached steady state in approximately 7 min. This effect was prevented by preloading the MCD with cholesterol. MCD-increased current was inhibited by low 4-aminopyridine concentration. Neonatal rat cardiomyocytes transfected with Green Fluorescent Protein (GFP)-tagged Kv1.5 channels showed a large ultrarapid delayed-rectifier current (I(Kur)), which was also stimulated by MCD. In atrial cryosections, Kv1.5 channels were mainly located at the intercalated disc, whereas caveolin-3 predominated at the cell periphery. A small portion of Kv1.5 floated in the low-density fractions of step sucrose-gradient preparations. In live neonatal cardiomyocytes, GFP-tagged Kv1.5 channels were predominantly organized in clusters at the basal plasma membrane. MCD caused reorganization of Kv1.5 subunits into larger clusters that redistributed throughout the plasma membrane. The MCD effect on clusters was sizable 7 min after its application. We conclude that Kv1.5 subunits are concentrated in cholesterol-enriched membrane microdomains distinct from caveolae, and that redistribution of Kv1.5 subunits by depletion of membrane cholesterol increases their current-carrying capacity.

Figure 1. Effects of cholesterol depletion on outward currents in native adult atrial myocytes

A, direct and long period effects of MCD application on the outward current elicited as indicated by the command waveform at 0.2 Hz, shown in the inset. B, effects of MCD–cholesterol application. C, time course changes in I_kur amplitude in control conditions, upon application of MCD and MCD–cholesterol complexes. D, bar graphs summarizing the effects of MCD and MCD–cholesterol complex, measured after 700 s application.

Figure 2. Pharmacological properties of the MCD-activated current in native adult atrial myocytes

Effects of 50 μm (A) and 2 mm 4-AP (B), and 10 mm TEA (C), on the 650 s MCD-stimulated outward current elicited as indicated by the command waveform at 0.2 Hz.

Figure 3. MCD effects on electrophysiological parameters of the outward K⁺ current of native adult atrial myocytes

Current traces elicited during incremental depolarizing test pulses in control (A) conditions and after MCD application (B). Cell capacitance: 75 pF. C, current density–voltage relationships of I_kur recorded in control conditions (•) and following MCD (^) application. D, voltage dependence of current activation in control (•) and MCD (^) conditions. In C and D, each point is the average of 7 cells.

Figure 4. MCD enhances Kv1.5-encoded currents in neonatal cardiomyocytes

A, traces of currents recorded during incremental 10 mV step depolarizations in control and in Kv1.5-transfected cardiomyocytes. B, effects of MCD on the outward current elicited by a test pulse from −80 to +60 mV in control, and after 350, 500 and 600 s MCD applications. Effect of 500 μm 4-AP on 650 s MCD-stimulated current is also shown. C, time course of the MCD and MCD–cholesterol complex effects on the outward current.

Figure 5. Effect of cholesterol depletion on outward current parameters resulting from Kv1.5 subunit overexpression in neonatal cardiomyocytes

Current density–voltage relationships (A) and voltage dependence (B) of I_Kur activation under control conditions (•) and following 7 min MCD application (^). In A and B, each point represents average data from 5 cells.

Figure 6. Kv1.5 do not co-localized with caveolin-3 in adult atrial tissue

Immunolocalization of Kv1.5 subunits (A) and caveolin-3 (B) in cryosections of atrial myocardium. Double immunostaining of connexin-43 (FITC, C) and caveolin-3 (Texas Red, D) in cryosections of atrial myocardium. E, merged image of the same area of the section in C and D, showing the lack of overlap between connexin-43 and caveolin-3 stainings.

Figure 7. Some Kv1.5 channel subunits are localized in lipid raft fractions in adult rat atrial myocytes

Western blot analysis of the distribution of Kv1.5 subunits, connexin-43 and caveolin-3 on step sucrose gradient of proteins from atrial myocardium.

Figure 8. Surface expression of Kv1.5 subunits in neonatal cardiomyocytes

A, in live cardiomyocytes, transfected GFP-tagged Kv1.5 subunits are clustered at the membrane surface adjacent to the bottom of laminin-coated glass support, as shown in the projection of Z sections in the lower panel. In contrast, GFP alone was homogeneously distributed in cardiomyocytes (inset). B, after the application of 2% MCD, clusters increased in size and were redistributed throughout the plasma membrane. C, bar graphs summarizing changes in cluster size upon MCD exposures; data are from 21 cardiomyocytes in control, and following incubation with 2% MCD for 7 min and 1 h 30 min. **P < 0.01, ***P < 0.001. D, double immunostaining of fixed cardiomyocytes using sarcomeric α-actinin and anti-Kv1.5 antibodies showing that Kv1.5-GFP-transfected cells are cardiomyocytes. A, B and D: scale bars represent 10 μm.