In this study we present 3 families with malignant hyperthermia (MH), all of Indian subcontinent descent. One individual from each of these families was fully sequenced for RYR1 and presented with the non‐synonymous change c.11315G>A/p.R3772Q. When present in the homozygous state c.11315*A is associated with myopathic symptoms. Muscle Nerve, 2009

Detecting and reacting to acute perturbation in the partial pressure of atmospheric oxygen (pO2), particularly hypoxia, is a fundamental adaptive mechanism which is conserved throughout the animal kingdom. In mammals, a number of cellular systems respond, often co-operatively as oxygen availability becomes compromised, with the express aim of maximising oxygen uptake by the lungs and of optimising its delivery to the metabolically most active tissues. Thus, during hypoxia, ventilation rate and depth are increased to maximize air flow across the gaseous exchange surface, local lung perfusion rates become rapidly matched to local alveolar ventilation and systemic arteriolar dilatation ensures that tissue and cerebral blood flow become swiftly optimized.

Recombinant and native large conductance, Ca(2+)-activated K+ (BK) channels often demonstrate O2 sensitivity in cell-free membrane patches suggesting that a significant component of the O2-sensing machinery must be closely associated with the channel protein complex. Until recently, however, the identity of the O2 sensor itself had remained elusive. Employing functional proteomics we have defined the molecular nature of such an O2 sensor of BK channels. Using immunoprecipitation, 1D and 2D gel electrophoresis, and mass spectroscopy we identified the constitutive form of haem oxygenase, haem oxygenase 2 (HO-2), as a BK alpha-subunit protein partner. Functional measurement of hypoxic modulation of BK channel activity during manipulation of HO-2 enzyme substrates and reaction products, followed by protein knock-down of HO-2 using small interfering RNA, indicated that this enzyme is directly involved in hypoxic inhibition of BK channels. Furthermore, good correlation was observed between data obtained from recombinant BK channels and those from acutely isolated rat carotid body glomus cells, suggesting strongly that HO-2 also acts as an O2 sensor in native arterial chemoreceptors.

This chapter assesses the use of stable cell lines for oxygen sensing by human recombinant K+ channels. Central to the cardiorespiratory responses to reduced O2 availability of all chemosensory tissue is the rapid inhibition of ion channels by hypoxia. Acute hypoxic modulation of K+ channel activity is central to chemosensing in the carotid body, neuroepithelial body, and its immortalized cellular counterpart (H146cells). In this study, the impact that recombinant human K+ channel expression systems have on the understanding of acute O2 chemotransduction by native and model chemosensory tissues is discussed. Different aspects related to generating stable cell lines, including the choice of cell line, cell culture, choice of vector, ligation of cloned channels into pcDNA3.1-TOPO, transfection and selection, verification of expression by RT-PCR, verification of expression by immunocytochemistry, and functional screening using the patch-clamp technique are discussed. . Homomultimeric O2-sensitive human K+ channels stably expressed in HEK293 cells and alternative approaches to the study of heteromultimeric O2-sensitive human K+ channels are also described.

Modulation of calcium-sensitive potassium (BK) channels by oxygen is important in several mammalian tissues, and in the carotid body it is crucial to respiratory control. However, the identity of the oxygen sensor remains unknown. We demonstrate that hemoxygenase-2 (HO-2) is part of the BK channel complex and enhances channel activity in normoxia. Knockdown of HO-2 expression reduced channel activity, and carbon monoxide, a product of HO-2 activity, rescued this loss of function. Inhibition of BK channels by hypoxia was dependent on HO-2 expression and was augmented by HO-2 stimulation. Furthermore, carotid body cells demonstrated HO-2–dependent hypoxic BK channel inhibition, which indicates that HO-2 is an oxygen sensor that controls channel activity during oxygen deprivation.

Large conductance, Ca2+-sensitive potassium (BK) channels are critical components of the O2 signalling cascade in a number of cells, including the carotid body and central neurones. Although the nature of the BK channel O2 sensor is still unknown, evidence suggests redox modulators might form part of the O2 sensing channel complex. By metabolising glutathione, γ-glutamyl transpeptidase (γGT) could act as such an O2 sensor. Western blotting and immunocytochemistry revealed high γGT expression in HEK293 cells expressing the α- and β-subunits of human recombinant BK and γGT co-immunoprecipitated with BKα. Acivicin blockade of γGT reversibly inhibited BK channels, suggesting that this BKα protein partner contributes to tonic channel activity. However, knock-out of γGT using siRNA had no effect on hypoxic BK channel inhibition. Together, these data indicate that γGT is a BKα protein partner, that its activity regulates BK channels but that it is not the BK O2 sensor.