Kinetic and thermodynamic study of a non-inactivating K+ current in a gonadotroph cell-line

Abstract

aT3-1 is a gonadotroph cell line derived from mice by oncogenic transformation of the pituitary, using simian virus, and secretes a subunit of follicle-stimulating hormone in response to hypothalamic peptide gonadotropin-releasing hormone. This thesis embodies studies on the detailed kinetic characterization of a non-inactivating K? current using the Hodgkin-Huxley (HH) model at low intracellular free Ca²? concentration. The thermodynamic parameters associated with channel activation were estimated from activation kinetics studied at different temperatures.

Chapter 1 focuses on the developments in electrophysiological methods and the revolutionary work of Hodgkin and Huxley. It lists out the possible sources of errors which can contaminate the time course of current waveforms and thereby the kinetics of a channel in voltage-clamp recording. It includes artifacts arising due to series resistance error, potassium accumulation, and digital leak subtraction. The kinetic characterization described in Chapter 4 was done using the HH formalism, and the thermodynamic analysis of channel activation described in Chapter 5 was done using the transition state theory. The principles associated with these analyses are discussed. The chapter also gives a general account of the diversity of K? channels and the K? channel gene family.

Chapter 2 is sub-divided into three broad headings: A) Tissue culture, B) Patch-clamp, and C) Tools for analysis and kinetic characterization of the ion channels using the HH model with inclusion of delay term.

Chapter 3 deals with electrophysiological and pharmacological methods in separating the various current components present in the membrane of aT3-1 cells. Isolated Na? and K? currents were recorded using appropriate recording solutions. aT3-1 cells were found to have only one type of sodium current but at least two types of K? currents in the presence of low free intracellular Ca²? concentration. Recordings from a holding potential (V_hold) of -80 mV showed that one of the current types was a transient K? current, whereas the other component was a non-inactivating K? current which activated at very depolarized potentials (>+40 mV). The transient current was blocked by 3 mM 4-amino-pyridine (4-AP) in the bath, but the non-inactivating K? current was not affected at similar concentrations of 4-AP. The transient K? current was completely inactivated by a 3-second long conditioning voltage-pulse positive to -15 mV. Depolarizing potentials from V_hold = -10 mV gave rise to non-inactivating K? currents, which were blocked by Charybdotoxin. The activation curve shifted towards negative potential with an increase in Ca²? concentration. The non-inactivating K? current recorded in the presence of 4-AP or from V_hold = -10 mV was concluded to be from a pure population of channels. This current was K? selective, and the activation time course was sensitive to free intracellular Ca²? concentration.

Chapter 4 presents an analysis of the non-inactivating K? current using the HH formalism. The HH equation was modified to incorporate the instrument delay and a free exponent in the fit. The steady-state activation plots of the non-inactivating K? current from V_hold = -80 mV in the presence of 3 mM 4-AP, or from V_hold = -10 mV, showed activation of current at potentials beyond +40 mV. For a 10°C increase in temperature, the activation plot showed a leftward shift of 4 mV. The time course of the current was fit with the equation:

I(t)=Imax(1?exp?(?(t?dt)?)) I(t)=I max ?

(1?exp(? ? (t?dt) ?

))

where dt is the instrument delay, and the exponent at is a free parameter. The value of at was close to 1 at all potentials at three different temperatures (15°C, 25°C, and 35°C). The results of the fit were used to estimate the rate constants of channel activation. The results support a simple two-state model for the channel kinetics, as shown in Scheme 1, with a single rate-limiting conformational change.

Scheme 1 CLOSE ? OPEN

While the activation threshold of the channel was weakly dependent on temperature, the rate constants for channel opening (?) and closing (?) were affected by temperature. The kinetics of the ensemble-averaged single-channel current mimicked the whole-cell current. The whole-cell current kinetics of the channel was similar to the cloned mslo K channel. Observations on the change in rate constants with temperature were used to estimate the thermodynamic parameters associated with channel activation.

Chapter 5 deals with the thermodynamics of K? current activation. The Arrhenius plot of forward and backward rates, obtained from the temperature-dependent study of non-inactivating K? currents, indicated the presence of a 10 kcal energy barrier between the closed and open states. The change in Gibbs free energy between open and closed states was estimated from: a) the plot of steady-state activation, and b) the difference in the transition state Gibbs free energy for backward and forward transitions. The estimate of Gibbs free energy from the two methods was in agreement. Variation of Gibbs free energy with potential showed a quadratic dependence on membrane potential, which was prominent at low temperatures of 15°C and 25°C; however, no significant deviation from a linear relation was noticed for the data at 35°C. At low temperature, the conformational changes in the channel molecule, subsequent to movement of the voltage-sensing element(s), were associated with the quadratic variation in Gibbs free energy with potential

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