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Comparison of drug blockade of a neuronal calcium-activated potassium channel with cardiac repolarizing potassium channels by potential class III agents

University of British Columbia

The aim of the study was to examine the use of a repolarizing calcium dependent potassium current K(Ca) in neurons to describe the actions of a group of novel compounds with potential Class ifi actions on cardiac cells. This was accomplished by determination of the correlation between potency of the agents to block single channel K(Ca) and potency to prolong effective refractory period (ERP) in heart. If a positive correlation could be established then elucidation of mechanisms of drug actions on single channel K(Ca) could have utility in the description of drug actions on repolarizing K+ currents in heart. At present the low unitary conductance of transient outward and delayed rectifier K+ channels precludes a mechanistic analysis of drug actions on cardiac cells. Initial experiments included the measurements of the single channel properties of the K(Ca) using inside-out patches obtained from cultured hippocampal neurons. The channel conductances, with physiological (SK+ and 140K+ across patches) and symmetrical (140 K+ across patches) were 110 pS and 170 pS respectively. A requirement of 4 µM internal calcium was necessary to maintain maximal channel activity with a threshold for K(Ca) activation at 0.7 µM. At low internal calcium concentration, depolarization increased the probability of channel openings. The effect was found to be solely dependent on the voltage-sensitive increase of the channel opening frequency; mean open times of the channel were not dependent on patch potential. The unitary K(Ca) is the microscopic basis for the macroscopic repolarizing current Ic in hippocampal neurons. Ic is responsible for the late repolarization phase and the early afterhyperpolarization (AHP) phase associated with the neuronal action potential. It was of interest to first determine the effects on Ic of an agent, tedisamil, with known Class ifi activity in heart. The results showed the drug both prolonged the neuronal action potential and eliminated the subsequent AHP phase. The primary set of experiments involved the investigations of the effects of 18 RSD novel compounds on unitary K(Ca). All the compounds, except for three, were effective in exhibiting rapid transitions in the K(Ca) from the opening state to a non-conducting state in the inside-out patches. The mean open times of open events were reduced but the closed time durations and the channel amplitudes were not changed. The potency of the effect of the RSD compounds on the K(Ca) was determined as the concentration required to halve the mean open time relative to control value. According to this index the compounds were categorized into five different groups based on potency to decrease mean open time of K(Ca). In addition the actions of five of the RSD compounds on K(Ca) were examined using outside-out patches excised from neurons. The potency of the RSD compounds on the neuronal K(Ca) was compared with their potency in inhibiting repolarizing K+ currents in the rat whole heart. The index for potency used in the whole heart experiments was the concentrations of the compounds required to increase the effective refractory period by 25%. Of the 18 compounds tested, 3 were found to be inactive (no obvious effect for concentration below 5OµM) on properties ofK(Ca). These same 3 agents also were ineffective in whole heart (in excess of 2OpM). For 13 of the remaining 15 agents, a positive correlation was found (with a correlation coefficient r of 0.71) between potency to block K(Ca) and potency to prolong ERP in whole heart. However, with 2 agents there was no apparent correlation for actions in the neurons and the heart. It was also established that drugs with persistent effects on K(Ca) (likely due to prolonged bonding to membrane sites) were also long-lasting in whole heart experiment.

Thesis/Dissertation

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2009-03-05

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http://hdl.handle.net/2429/5546

Pharmacology

University of British Columbia

UBCV

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