Silencing vascular smooth muscle ATP-sensitive K⁺ channels with caveolin-1

ATP-sensitive K⁺ (K_ATP) channels are octameric complexes consisting of four pore-forming subunits of the Kir6 inwardly rectifying K⁺ channel subfamily (i.e. Kir6.1 and/or Kir6.2), and four regulatory sulfonylurea receptor (SUR) subunits that are members of the ATP-binding cassette family of proteins (i.e. SUR1, SUR2A or SUR2B) (Bryan et al. 2004; Teramoto, 2006; Zhang et al. 2009). Traditionally, these potassium channels have been viewed as the link between cellular metabolism and electrical excitability, with reductions in intracellular ATP/ADP ratio being the stimulus for increased channel opening (gating), membrane potential hyperpolarisation and suppression of action potential initiation in conditions of low metabolic activity. ATP binding to the Kir6 subunits is inhibitory, but this is offset by the activating influence of MgADP interacting with the SUR subunits (Bryan et al. 2004; Teramoto, 2006; Zhang et al. 2009). Recent findings indicate, however, that the ATP/ADP ratio may not be the physiological regulator of K_ATP channel gating (Zhang et al. 2009). Rather, accumulating evidence implies that the nucleotide sensitivity of the channels is modulated, for example by alterations in membrane levels of PIP₂ and fatty acyl CoA esters, as well as phosphorylation by protein kinases (Zhang et al. 2009). In this context, the K_ATP channels expressed in vascular smooth muscle cells are of particular interest, as they exhibit activity under normal physiological conditions and contribute to the control of membrane potential, arterial diameter and blood flow regulation (Teramoto, 2006). The paper by Davies et al. (2010) in this issue of The Journal of Physiology provides new insights concerning the modulation of vascular smooth muscle K_ATP channel gating by caveolin-1 that may have important implications for the control of diameter and blood flow in health and disease.

The major K_ATP channel isoforms expressed in cardiac ventricular myocytes, pancreatic β-cells and vascular smooth muscle cells are composed of Kir6.2/SUR2A, Kir6.2/SUR1 and Kir6.1/SUR2B subunits, respectively (although other isoforms with different subunits may also be present; Teramoto, 2006; Zhang et al. 2009). These channel subtypes exhibit distinct biophysical properties (e.g. single channel conductance, pattern of gating behaviour, and/or extent of inward rectification) and sensitivities to ATP and MgADP (Teramoto, 2006). Vascular smooth muscle K_ATP channels exhibit a distinct bell-shaped sensitivity to intracellular MgADP, with physiological levels (0.1–1 mm) being sufficient to stimulate gating (Zhang & Bolton, 1996). Also, vascular smooth muscle K_ATP channel activity is modulated by cellular signalling pathways in response to G protein-coupled receptor activation. Vasodilators such as calcitonin gene related peptide and adenosine increase, whereas vasoconstrictors including angiotensin II and endothelin-1 inhibit gating via protein kinase A and C signalling pathways, respectively (Sampson et al. 2004, 2007; Teramoto, 2006). This regulation appears to be facilitated by the scaffolding protein, caveolin-1, that mediates the co-localisation of K_ATP channels with elements of these signalling pathways in cholesterol-enriched caveolar membrane domains (Sampson et al. 2004, 2007). Binding to caveolin was suggested to reduce K_ATP current amplitude in cultured vascular smooth muscle cells by promoting channel internalisation (Jiao et al. 2008), but the effect on gating or nucleotide sensitivity was not considered.

Davies et al. now make the novel observation that the interaction of recombinant Kir6.1/SURB and native aortic K_ATP channels with caveolin-1 suppresses gating by reducing the sensitivity of channels to the stimulatory influence of MgADP. Davies et al. first demonstrated that whole-cell Kir6.1/SUR2B current density in HEK 293 cells was larger in the absence of caveolin-1, and reduced by intracellular application of a caveolin-1 scaffolding domain peptide. Significantly, the scaffolding domain peptide was also shown to suppress K_ATP current of freshly isolated rat aortic myocytes, indicating that the phenomenon is relevant to channels in a native cellular environment. The decrease in current in these experiments could be due to a reduction in the number of channels at the cell surface by internalisation (Jiao et al. 2008), or alternatively, a change in gating or single channel conductance. Davis et al. distinguish between these possibilities by studying the effect of caveolin-1 on nucleotide sensitivity and the pattern of single channel gating. The bell-shaped curve for MgADP modulation of channel gating was left-shifted, such that the sensitivity to ADP over the physiological concentration range was reduced in the presence of caveolin-1. Gating of vascular K_ATP channels is characterised by bursts of rapid, flickery openings and closings separated by long interburst periods of channel closure (Zhang & Bolton, 1996). Davies et al. found that the interaction of Kir6.1/SUR2B channels with caveolin-1 was associated with a dramatic increase in the length of the interburst interval that was best explained by an increase in transition into the interburst closed state and fewer transitions between two open states during the bursts. These findings are consistent with the idea that caveolin modulates K_ATP channel gating and imply that this important scaffolding protein should be regarded as novel factor that regulates K_ATP channel nucleotide sensitivity.

Despite the wealth of information that has been generated since K_ATP channels were first described over twenty years ago, we remain ignorant of many aspects concerning their complex regulation and physiological function. This is especially true for vascular smooth muscle K_ATP channels compared to the isoforms of β-cells and cardiac myocytes that grab the vast majority of headlines. The findings of Davies et al. advance our understanding of the regulation of vascular K_ATP channels, but they also raise several new issues. For example, the molecular mechanism by which caveolin-1 elicits the change in gating is not defined. MgADP is known to interact with the second nucleotide binding fold of the SUR subunit to stimulate K_ATP channel gating (Bryan et al. 2004; Teramoto, 2006; Zhang et al. 2009). Davies et al. note that there are two potential caveolin binding sites in SUR2B and none in Kir6.1. Does this mean that the interaction with caveolin-1 interferes with MgADP binding to the SUR subunit, and if so, is the mechanism similar to the interaction of syntaxin-1A with nuclear binding folds of SUR2A (Kang et al. 2004)? Secondly, does the interaction of caveolin-1 represent a mechanism for short-term, reversable regulation of gating, or is it simply a prelude to channel internalisation and degradation? Thirdly, is the effect of caveolin on gating and MgADP sensitivity shared by the K_ATP isoforms expressed in cardiac myocytes and β-cells? And finally, is the regulation of K_ATP gating by caveolin-1 relevant to the control of vascular smooth muscle membrane potential and blood flow in vivo? This latter point is particularly important. Davies et al. speculate that increased caveolin-1 expression in pathophysiological situations of elevated cholesterol may ‘silence’ populations of vascular smooth muscle cell K_ATP channels leading to depolarisation, increased sensitivity to vasoconstrictors, reduced arterial diameter and hypertension. Whether the mechanism identified by Davies et al. using an over-expression system and conduit vessel myocytes is relevant to the development of dysfunctional contractility in the small arteries/arterioles that determine peripheral arterial resistance and regulate blood flow is a key issue that awaits confirmation. Despite these limitations, the findings of Davies et al. make a strong argument for considering caveolin-1 as a modulator of K_ATP channel nucleotide sensitivity and gating, as well as providing a stimulus for future studies to assess the relevance and physiological significance of the mechanism within and outside of the vasculature.