Program Objectives:

             The objective of this research program is to characterize mechanisms regulating voltage-dependent K⁺ (Kv) channel function and interaction with the secretory machinery in endocrine cells. This program has three related aims: to study i) the regulation of Kv2.1 channels by metabolic signals; ii) the regulation of Kv2.1 channels by covalent peptide modification; and iii) the interaction between Kv2.1 and the exocytotic SNARE proteins. Our current focus is on the second Aim.

 

             ii) regulation of Kv2.1 function by covalent peptide modification. This is a project that has been recently initiated in our new laboratory. Recent exciting work^3;4 demonstrates that covalent peptide modification regulates the function of membrane leak K⁺ and Kv1.5 ion channels. Specifically, this was due to the covalent attachment of small ubiquitin like modifier (SUMO) proteins, through a process known as SUMOylation. Kv2.1 contains three potential intracellular SUMOylation motifs, and preliminary data from our laboratory demonstrates the expression of SUMO isoforms 1, 2 and 3 in insulin secreting cells, and indicate that direct infusion of SUMO1 increases native Kv current in insulin secreting cells. It is unclear at this point whether this results from increased SUMOylation of the channel or whether the infused SUMO1 peptide acts in a ‘dominant-negative’ manner (i.e. to prevent SUMO-dependent interaction with regulatory proteins). I think this will prove to be a very interesting area of study.

            

Literature Review:

 

III.a)The voltage-dependent K⁺(Kv) channels: There are 11 mammalian Kv channel families and various related families known.⁷ Kv channels are formed by the tetrameric assembly of 6-transmembrane domain a-subunits which co-assemble as hetero-tetramers in a family specific manner.⁸ Some of these associate with cytosolic or trans-membrane regulatory subunits. Kv currents are often classified based on their biophysical properties. A-currents activate and inactivate quickly upon a step membrane potential depolarization, giving rise to the characteristic waveform for which reason they are named. Delayed-rectifier currents activate more slowly and do not inactivate (or inactivate slowly over seconds). Kv channel isoforms have well established roles in excitable cells including neurons,^9;10 muscle cell types,^2;11-13 and secretory cells.¹⁴ Kv channels are critically important modulators of electrical activity and are activated by membrane depolarization (usually in the up-stroke of an action potential), mediate an outward K⁺ current, and re-polarize action potentials. Kv channels are also abundantly expressed in non-excitable cells such as epithelial cells⁹ and cells of the immune system^15;16, where they may play a role in regulating proliferation. Our main interest is in the regulation of secretory processes in insulin producing b-cells of the pancreas. Secretion of insulin from these cells is critically dependent on membrane potential depolarization, action potential firing, and entry of Ca²⁺ through voltage-dependent Ca²⁺ channels (VDCCs).¹⁷ Thus, inhibition of Kv currents in b-cells leads to action potential prolongation, increased intracellular Ca²⁺ ([Ca²⁺]_i), and increased insulin secretion.¹⁸ My previous work has identified the Kv2.1 isoform (a delayed rectifier channel that is homologous to the drosophila Shab channel) as the major contributor to b-cell Kv currents and a regulator of insulin secretion.^1;5;18-22

 

III.b)Metabolic regulation of Kv2.1: Signals from mitochondrial metabolism are important regulators of cellular excitability. The best-studied is ATP which acts to close ATP-sensitive K⁺ channels, depolarize the b-cell, and thus trigger insulin secretion. Recent studies have suggested that mitochondrial NADPH production by pyruvate cycling,^23-25 might be an important metabolic signal in insulin secreting cells. Indeed, metabolisable insulin secretagogues increase the NADPH:NADP⁺ ratio in rodent islets^26;27 and inhibition of NADPH formation reduces glucose-stimulated insulin secretion.^28;29 Recent evidence suggests the existence of membrane associated aldehyde oxidoreductase-like enzyme activity in rat islets³⁰ and a number of oxidoreductase-like Kv b-subunits (Kvb1, 2 and 3- cytosolic Kv regulatory proteins) are expressed in insulin secreting cells.³¹ Recently, we have described a potent regulation of native Kv2.1 channels in b-cells by cytoplasmic NADPH,¹ wherein raising the NADPH/NADP⁺ ratio caused b-cell Kv2.1 currents to inactivate quickly and more completely, and a leftward shift in the voltage-dependence of steady-state inactivation (meaning that more channels were already inactivated). This has important implications since the metabolic generation of NADPH could reduce the efficacy of Kv channels in repolarising the b-cell.

 

III.c) Regulation of ion channels by SUMOylation: The small ubiquitin-like modifier (SUMO) peptides can be covalently linked to proteins, including ion channels.^3;4 However unlike ubiquitin attachment which targets proteins toward degradation pathways, SUMOylation and de-SUMOylation of specific target motifs (by SUMO-specific ligases and proteases) modifies protein function by regulating interaction with secondary proteins.^32;33 There are at least 4 mammalian SUMO isoforms (SUMO-1, 2, 3 and 4), of which isoforms 1-3 are ubiquitously expressed (including in insulin secreting cells- our preliminary results), whereas isoform 4 appears restricted to immune cells and kidney. Recently, SUMOylation of a Kv channel (Kv1.5) was shown to cause a 15 mV hyperpolarizing shift in the steady-state voltage-dependence of inactivation.³ This means that at any given membrane potential, more Kv channels are already inactivated and thus unavailable for action potential repolarization (this could be expected to broaden action potentials). Our analysis of the Kv2.1 protein sequence predicts 3 (rat, mouse) or 4 (human) potential intracellular SUMOylation sites (based on SUMOylation site prediction³⁴).

 

III.d) Kv channels associate with the SNARE complex: SNARE (soluble NSF attachment receptor) proteins constitute the molecular machinery regulating vesicle docking and fusion. Vesicle-associated SNARE proteins include the VAMPs (or synaptobrevins). SNARE proteins associated with the target membrane (or t-SNAREs) include SNAP-25 and syntaxin. While the SNARE complex mediates the molecular events of exocytosis upon elevation of [Ca²⁺]_i, it is also functionally coupled to ion channels in a unit termed the 'excitosome'. The t-SNARE proteins interact with and regulate the function of voltage-dependent Ca²⁺ and Kv channels.^5;35-37 We recently reported that SNAP-25 and syntaxin 1A can bind Kv2.1 and that SNAP-25 can inhibit recombinant Kv2.1 by 70% and native Kv2.1 in b-cells by 40% through an interaction with the Kv2.1 N-terminus,⁵ although the biophysical mechanism of this effect was not studied. However, it seems clear that SNARE proteins have an important role in the regulation of excitability, allowing feedback between exocytosis and excitability. More recent work has shown that the interaction between Kv2.1 and syntaxin has an important positive role in exocytosis per se, independent of the ability of Kv2.1 to conduct current.⁶ This finding suggests that the importance of Kv2.1 in secretion may extend beyond its role in membrane repolarization.

 

 

Anticipated Significance:

             Voltage-dependent K⁺ channels have important roles regulating cellular excitability. Our understanding of the mechanisms regulating these is however incomplete. Metabolic and redox sensing by Kv2.1 may play an important role in physiological processes such as oxygen-sensing in the pulmonary vasculature and in oxygen-sensing neurons, and in the process of glucose-stimulated insulin secretion. Regulation of these channels by covalent peptide modulation is only beginning to be understood and likely plays an important role in the adaptive responses of neurons and smooth muscle for example. Understanding the mechanism by which SUMOylation affects Kv2.1 will provide insight into these processes. Finally, the prospect that these channels have a pivotal role in the docking and exocytosis of secretory granules, separate from their role in membrane excitability, is indeed interesting and may indicate a role for Kv2.1 in the ‘excitosome’ complex (with Ca²⁺ channels and the exocytotic machinery) thought to be important for neuro- as well as endocrine secretion. These studies will add to our fundamental understanding of the regulation of the Kv2.1 channel and its interaction with secretory processes.

 

References

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