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Electrophysiology of potassium channels in the hamster egg

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McNivenAI_1989redux.pdf (23.37Mb)
Date
1989
Author
McNiven, Alistair Iain
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Abstract
 
 
Ca²⁺-activated K⁺ currents were recorded from unfertilised and fertilised golden hamster eggs, using conventional intracellular recording techniques. Activation of the currents was accomplished by various treatments producing a rise in the intracellular free calcium concentration ([Ca²⁺]ᵢ;). The resulting hyperpolarisations of the membrane were investigated using Ca²⁺ extrusion blockers such as La³⁺, quercetin, 2,4-Dinitrophenol, high pH₀ solution and Na⁺-free solution, to alter the time-course of the hyperpolarisations.
 
Iontophoretic injection of Ca²⁺ and Sr²⁺ produced a transient hyperpolarisation of the hamster egg membrane accompanied by an increase in membrane conductance, mediated by a Ca²⁺-activated K⁺ current. The effects of La³⁺, quercetin, high pH₀ solution and Na⁺-free solution in prolonging the hyperpolarisation suggest that both a Na⁺-Ca²⁺ exchange system and an active Ca²⁺ pump are responsible for the recovery phase of the Ca²⁺-evoked hyperpolarisations.
 
A combination of high pH₀ and high [Ca²⁺]₀ solution was found to evoke a sustained hyperpolarisation of the membrane, with an estimated value of the reversal potential of about 85 mV. The membrane potential changed linearly with log [K⁺]₀ with a slope of 43 ± 2 mV (mean ± S.D., n=4) for a 10-fold change in [K⁺]₀, while it was unaltered by the removal of CI from the solution. The amplitude of the pH₀-induced hyperpolarisation decreased substantially when [Ca²⁺]₀ was lowered from 20 to 1 mM and with addition of TEA. Injection of EGTA into the egg prevented the pH₀-induced hyperpolarisation, suggesting that a rise in [Ca²⁺]ᵢ is required. The high pH₀ does not produce a large increase in pHᵢ and the onset of the response is rapid, compatible with an external site of action.
 
A transient hyperpolarisation and increase in conductance could be evoked after a long latency (ca. 9 s) by a single Ca²⁺ action potential in unfertilised hamster eggs. The estimated reversal potential was close to Ek. A second action potential elicited soon after the first did not induce a similar response. A number of treatments (insertion of a Ca²⁺ micro-pipette, aplication of Na⁺-free solution, La³⁺or high pH₀) likely to raise [Ca²⁺]ᵢ also induced similar large hyperpolarisations, after which a single Ca² action potential failed to evoke a large delayed hyperpolarisation. This suggests that a small rise in [Ca²⁺]ᵢ activates a slow process leading to a further large increase in [Ca²⁺]ᵢ.
 
The fertilisation potential of hamster eggs, consisting of transient, repetitive hyperpolarisations was recorded using intracellular recording techniques. The duration of hyperpolarisations were prolonged by La³⁺, high pH₀ and Na⁺-free solution. Low pH₀ decreased the duration. The frequency of the hyperpolarisations was also reduced by Co²⁺ and La³⁺.
 
The first single channel recordings from the hamster egg were made, using the excised inside-out patch conformation. Evidence of a channel activated by intracellular Ca²⁺ was obtained. This channel had a reversal potential at 0 mV in symmetrical KCl, and displayed outward rectification over the potential range - 60 - + 60 mV. The conductance was 65.3 ± 12.9 pS at depolarised potentials and 23.4 ± 11.8 pS (n=4) at hyperpolarised potentials.
 
URI
http://hdl.handle.net/1842/29885
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