Gut hypoxia during major surgery is associated with increased intestinal permeability, bacterial translocation, systemic sepsis and multi-organ failure. K⁺ channel activation represents a common response to metabolic inhibition in a number of different cell types. Acute metabolic stress produced by 2,4dinitrophenol (DNP) and deoxyglucose (DG) activates large conductance K⁺ channels in cardiovascular tissues, and small conductance K⁺ channels in liver and biliary cell lines. In the T₈₄ colonic adenocarcinoma cell line, mastoparan caused a 4-fold increase in paracellular permeability that was linked to increased basolateral membrane K⁺ conductance. Thus, modulation of K⁺ channel activity represents a potential target for reducing the risk of systemic sepsis due to hypoxic intestinal injury. The aim of the current study was to determine whether acute metabolic stress leads to activation of basolateral K⁺ channels in human colonic crypts. Biopsies of normal sigmoid colonic mucosa were obtained from patients undergoing routine colonoscopy. Crypts were isolated by Ca²⁺ chelation (Bowley et al. 2003). Whole-cell K⁺ currents were measured using the perforated patch-clamp technique (0.24mg/ml amphotericin in pipette), and single channel activity in the basolateral membrane was studied in the cell-attached configuration. Cells were exposed to 100μM DNP + 5mM DG to produce metabolic inhibition. Data are presented as mean ± 1 SEM, with n, the number of experiments, and were compared by Student’s paired t test. Metabolic inhibition stimulated whole-cell currents within 5 minutes, with an increase in whole-cell conductance from 1.45 ± 0.13nS to 3.29 ± 0.19nS (P<0.005; n = 11), and hyperpolarization of the cell membrane voltage from -67 ± 3mV to -80 ± 3mV (P<0.025), consistent with K⁺ channel activation; these effects were completely inhibited by the specific IK_Ca inhibitor, TRAM-34. In single channel recordings, metabolic inhibition increased IK_Ca activity; NPo, where N = number of channels and Po = open probability, increased from 0.58 ± 0.2 to 0.81± 0.3 (p<0.05, n=10) In conclusion, metabolic stress activates basolateral K⁺ channels (IK_Ca) in human colonic crypts, resulting in hyperpolarization of the membrane potential. Further studies are required to establish if IK_Ca stimulation is associated with an increase in paracellular permeabilility. If so established, specific K⁺ channel blocking drugs may be of theraputic use in the prevention of systemic sepsis associated with gut hypoxia.