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Using biophysical techniques, we previously have implicated outwardly rectifying potassium currents in the proliferation of cultured spinal cord astrocytes and have demonstrated that delayed rectifier potassium currents (IKd), in particular, are upregulated on entry into the cell cycle and downregulated with cell cycle exit and differentiation. In the present study, using specific antibodies and antisense oligodeoxynucleotides, we show that this proliferation-dependent potassium current is mediated by theShakerpotassium channel Kv1.5. Downregulation of Kv1.5 protein by antisense oligodeoxynucleotides reduces astrocyte proliferation by ∼50%, although no observed changes occur in Kv1.5 protein expression during spontaneous differentiation in culture. Tyrosine phosphorylation of Kv1.5, however, is downregulated markedly in differentiated cells but unaltered on cell cycle arrest. Using immunoprecipitation, we show that Kv1.5 is associated with Src family protein tyrosine kinases and that this association does not change with cell differentiation. Inhibition of kinase activity with the Src-specific inhibitor PP2 decreases Kv1.5 phosphorylation, reducesIKd, and inhibits astrocyte proliferation, specifically in the G0/G1phase of cell cycle. Conversely,IKdare potentiated when active Src is present in the pipette. Transfection of quiescent astrocytes with constitutively active Src (Src Y529F) causes marked upregulation of astrocyte proliferation. These data suggest that Kv1.5 is phosphorylated constitutively by Src kinases during growth and that downregulation of Src activity may underlie both astrocyte differentiation and the accompanying changes in delayed rectifier potassium channel activity.

Arrest of spinal cord astrocytes at defined stages of the cell cycle clock causes significant changes in the expression of voltage-activated Na+ and K+ currents. Arrest of actively proliferating astrocytes in G1/G0 by all-trans-retinoic acid induces premature expression of inwardly rectifying K+ currents (IKIR) typically expressed only in differentiated astrocytes. By contrast, arrest in S phase by ara-C or Aphidicolin leads to a greater than twofold increase in “delayed” outwardly rectifying currents (IKD) and a concomitant decrease in IKIR. Pharmacological blockade of IKD by TEA and 4AP caused proliferating astrocytes to arrest in G0/G1, suggesting that activity of these channels is required for G1/S checkpoint progression. Conversely, in quiescent astrocytes, inhibition of IKIR by 30 μM BaCl2 led to an increase in astrocyte proliferation and to an increase in the number of cells in S phase from 5% to 26%. These data suggest that a downregulation of KIR promotes cell cycle progression through the G1/S checkpoint. Blockade of IKIR in actively proliferating cells, however, leads to an accumulation in G2/M, suggesting that reappearance of this current may be critical for progression beyond DNA synthesis. Interestingly, Na+ currents (INa+) are increased greater than fourfold in S phase-arrested cells, yet their pharmacological blockade by TTX has no effect on cell cycle progression. However, the resting membrane potential of S phase-arrested cells increases profoundly, and manipulation of membrane potential by the application of low concentrations of ouabain, or reduction of extracellular potassium, induces the accumulation of quiescent astrocytes in S phase of the cell cycle, suggesting that either depolarization or intracellular sodium, or both, play an important role in promoting astrocyte proliferation. GLIA 30:39–48, 2000. © 2000 Wiley-Liss, Inc.

MacFarlane, Stacey Nee and Harald Sontheimer. Spinal cord astrocytes display a switch from TTX-sensitive to TTX-resistant sodium currents after injury-induced gliosis in vitro. J. Neurophysiol. 79: 2222–2226, 1998. Two distinct morphological subtypes of astrocytes have been shown to express Na+ currents that differ biophysically and pharmacologically. Using an in vitro model for reactive gliosis, we recently reported marked changes in Na+ and K+ channel expression by astrocytes induced to proliferate. Using this in vitro assay in which a confluent monolayer of astrocytes is mechanically scarred to induce gliosis, we now demonstrate that sodium currents of scar-associated cells, in addition to doubling in current density, also switch from being tetrodotoxin-sensitive(TTX-S, IC50 8 nM) to being ∼40-fold more TTX-resistant (TTX-R,IC50 314 nM). These changes occurred within 6 h after injury and were not associated with any notable changes in cell morphology. Changes in biophysical properties were analyzed for the two current types. The activation curve for TTX-R currents demonstrated a significant depolarized shift versus that of TTX-S currents ( P ≤ 0.003), and TTX-R currents have more depolarized V1/2 of activation (−33 vs. −23 mV). The V1/2 of inactivation was slightly, but not significantly, more depolarized for TTX-R currents as compared to TTX-S (−63 vs. −68 mV). Most notably, TTX-R currents showed significantly slower inactivation kinetics at depolarized voltage potentials than TTX-S sodium currents (0.76 vs. 1.128 ms, at −10 mV; P < 0.0004).

The functions of ion channels can be regulated by their phosphorylation state. Protein kinases and protein phosphatases tightly control the activity of channels, thereby regulating the flow of ions across cell membranes. Channel proteins and kinases or phosphatases can associate directly or through intermediate adaptor proteins. An interaction domain termed the leucine zipper (LZ), once thought to be unique to some families of transcription factors, has been identified in channel proteins and their cognate binding proteins. MacFarlane and Levitan discuss what roles LZ-containing proteins might have in controlling channel function.

Anin vitroinjury model was used to examine the electrophysiological changes that accompany reactive gliosis. Mechanical scarring of confluent spinal cord astrocytes led to a threefold increase in the proliferation of scar-associated astrocytes, as judged by bromodeoxyuridine (BrdU) labeling. Whole-cell patch-clamp recordings demonstrated that current profiles differed absolutely between nonproliferating (BrdU−) and proliferating (BrdU+) astrocytes. The predominant current type expressed in BrdU−cells was an inwardly rectifying K+current (KIR; 1.3 pS/pF). BrdU−cells also expressed transient outward K+currents, accounting for less than one-third of total K+conductance (G). In contrast, proliferating BrdU+astrocytes exhibited a dramatic, approximately threefold reduction in KIR(0.45 pS/pF) but showed a twofold increase in the conductance of both transient (KA) (0.67–1.32 pS/pF) and sustained (KD) (0.42–1.10 pS/pF) outwardly rectifying K+currents, with aGKIR:GKDratio of 0.4. Relative expression ofGKIR:GKDled to more negative resting potentials in nonproliferating (−60 mV) versus proliferating astrocytes (−53 mV;p= 0.015). Although 45% of the nonproliferating astrocytes expressed Na+currents (0.47 pS/pF), the majority of proliferating cells expressed prominent Na+currents (0.94 pS/pF). Injury-induced electrophysiological changes are rapid and transient, appearing within 4 hr postinjury and, with the exception of KIR, returning to control conductances within 24 hr. These differences between proliferating and nonproliferating astrocytes are reminiscent of electrophysiological changes observed during gliogenesis, suggesting that astrocytes undergoing secondary, injury-induced proliferation recapitulate the properties of immature glial cells. The switch in predominance from KIRto KDappears to be essential for proliferation and scar repair, because both processes were inhibited by blockade of KD.