Sherwin C. Lee

Voltage-gated potassium conductance in human T lymphocytes stimulated with phorbol ester.

The whole‐cell patch‐clamp method was used to study the voltage‐gated K+ conductance of human peripheral blood T lymphocytes. After entry into whole‐cell recording mode, there are time‐dependent changes in some properties of the conductance. Over the first 10‐30 min, the threshold for activation shifts about 10 mV more negative, and the rates of activation and inactivation increase. Inactivation is less strongly voltage dependent than activation or deactivation. Lymphocytes were stimulated to proliferate in culture with the tumour promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). No changes in K+ conductance were observed in the first few hours of TPA stimulation. At 24 h after mitogen addition, TPA‐treated cells were found to have 1.7‐fold greater average voltage‐gated K+ conductance than unstimulated control cells. At 48 h, TPA‐stimulated cells had the same average K+ conductance as at 24 h, even though the cells were now much increased in size, as measured by cell capacitance. DNA synthesis by cultures stimulated with TPA, phytohaemagglutinin or succinyl concanavalin A was depressed by the addition of 0.1 mM‐quinine at any point in the culture period. In the first 20 h after mitogen addition, DNA synthesis was more effectively inhibited by quinine than if the drug were added later. Cell proliferation was equally sensitive to quinine regardless of mitogen.

Temperature dependence of K(+)-channel properties in human T lymphocytes.

We have used whole-cell patch clamp to determine the temperature dependence of the conductance and gating kinetics of the voltage-gated potassium channel in quiescent, human peripheral blood T lymphocytes. Threshold for activation, steady-state inactivation, and the reversal potential are the same at 22 degrees and 37 degrees C. However, the time-constants for activation, inactivation, deactivation, and release from inactivation are quite sensitive to temperature, changing by at least a factor of five in each case over this range of temperatures. The onset of cumulative inactivation at 22 degrees and 37 degrees C reflects the time-course of deactivation. Peak outward current is approximately twofold greater at 37 degrees C than at 22 degrees C; this increase is also manifest at the single channel level. Energies of activation for conductance, activation, inactivation, deactivation, and release from inactivation are 8.2, 22.1, 25.0, 36.2, and 42.2 kcal/mol, respectively. No new channels were observed at 37 degrees C, and there was no evidence for alteration of the K+ conductance by putative modulators at 22 or 37 degrees C.

Cell Volume Regulation in Lymphocytes

This article reviews what is known about the volume regulatory responses of lymphocytes. We present a discussion of recent data and hypotheses pertaining to the underlying mechanisms in regulatory volume increase (RVI) and regulatory volume decrease (RVD). New results from our laboratory are included to demonstrate that RVD is modulated by both temperature and pH, and that RVD occurs in proliferating as well as quiescent lymphocytes. This information is considered in the context of a model that includes the dynamics of membrane potential, K+ conductance. Cl- conductance, a proposed stretch-activated conductance, gating mechanisms, and equilibrium potentials, as RVD progresses. The physiological relevance of volume homeostasis in lymphocyte function, in particular, and in cell growth and proliferation, in general, is discussed.

Modulation of K+ currents in human lymphocytes by pH.

1. Using whole‐cell patch‐clamp techniques, we found that the voltage‐dependent K+ conductance in human peripheral blood T lymphocytes is enhanced threefold at alkaline intracellular pH (pHi) compared to acid pHi. This pH dependence can be described by a model having two strongly co‐operative proton binding sites with pka 7.15. A similar pHi sensitivity exists for K+ conductance in mitogen‐activated cells. 2. The reversal potential, threshold voltage for activation of the K+ conductance, and voltage dependence of steady‐state inactivation are not affected by pHi. Activation and inactivation kinetics are also unchanged. 3. Single‐channel measurements made in whole‐cell patch‐clamp mode indicate that the effect of intracellular pH on the amplitudes of single‐channel events parallels, but does not wholly account for, the effect of pHi on the macroscopic currents. 4. Lowering extracellular pH (pHo) shifts the threshold for activation of the K+ current to a more depolarized voltage, consistent with a surface charge screening effect. Apparent changes in peak current and activation kinetics at acid pHo can be accounted for by this voltage shift. An additional slowing of inactivation kinetics at low pHo does occur. 5. The relevance of the pH sensitivity of the voltage‐gated K+ conductance to lymphocyte mitogenesis and volume regulation is discussed.

Forskolin effects on the voltage-gated K+ conductance of human T cells

Forskolin, a direct activator of adenylate cyclase, modifies the voltage-dependent K+ conductance of quiescent human peripheral blood T lymphocytes. In the presence of greater than 20 μM forskolin, the average voltage-gated current in whole-cell patch clamp is significantly decreased. The voltage dependence and kinetics of activation are not changed from untreated control cells. However, inactivation becomes biphasic. Much of the current inactivates very quickly (complete in 10 ms), and the remaining outward current inactivates more slowly with a time constant closer to that of control cells. To determine whether this effect is mediated by a rise in intracellular cAMP, cells were preincubated and subsequently voltage-clamped in the presence of other agents that raise the cAMP levels in T cells (isoproterenol plus a phosphodiesterase inhibitor, or dibutyryl cAMP) with no effect on the K+ conductance. Similarly, cells put in whole-cell patch clamp with cAMP, GTP, ATP, and theophylline added to the electrode filling solution showed no change in K+ current. Because other proccdures that raise cAMP did not duplicate the effect of forskolin, we investigated the effect of 1,9-dideoxyforskolin, an analogue of forskolin that does not stimulate adenylate cyclase in human lymphocytes. This drug induced changes in the whole-cell K+ conductance identical to those observed with forskolin. Both forskolin and dideoxyforskolin inhibit mitogen-induced proliferation of lymphocytes. Because inhibition of proliferation occurs in the presence of known K+ channel blockers, these results suggest that forskolin has an effect on T cell mitogenesis that is mediated by inhibition of K+ conductance and is independent of cAMP.